An evolutionary view on thylakoid protein phosphorylation uncovers ...

REVIEW PAPER

An evolutionary view on thylakoid protein phosphorylation uncovers novel phosphorylation hotspots with potential functional implications

Michele Grieco1, Arpit Jain2, Ingo Ebersberger2,3 and Markus Teige1,*1 Department of Ecogenomics and Systems Biology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria2 Department for Applied Bioinformatics, Institute for Cell Biology and Neuroscience, Goethe University, Max-von-Laue Str. 13, D-60438 Frankfurt, Germany3 Senckenberg Biodiversity and Climate Research Centre (BiK-F), Senckenberg Anlage 25, D-60325 Frankfurt, Germany

* Correspondence: [email protected]

Received 12 February 2016; Accepted 5 April 2016

Editor: Christine Raines, University of Essex

Abstract

The regulation of photosynthetic light reactions by reversible protein phosphorylation is well established today, but functional studies have so far mostly been restricted to processes affecting light-harvesting complex II and the core proteins of photosystem II. Virtually no functional data are available on regulatory effects at the other photosynthetic complexes despite the identification of multiple phosphorylation sites. Therefore we summarize the available data from 50 published phospho-proteomics studies covering the main complexes involved in photosynthetic light reac-tions in the ‘green lineage’ (i.e. green algae and land plants) as well as its cyanobacterial counterparts. In addition, we performed an extensive orthologue search for the major photosynthetic thylakoid proteins in 41 sequenced genomes and generated sequence alignments to survey the phylogenetic distribution of phosphorylation sites and their evolu-tionary conservation from green algae to higher plants. We observed a number of uncharacterized phosphorylation hotspots at photosystem I and the ATP synthase with potential functional relevance as well as an unexpected diver-gence of phosphosites. Although technical limitations might account for a number of those differences, we think that many of these phosphosites have important functions. This is particularly important for mono- and dicot plants, where these sites might be involved in regulatory processes such as stress acclimation.

Keywords: Acclimation to stress, calcium signal, chloroplast, evolution, light-harvesting, photosynthesis, protein phosphorylation, signalling.

Protein phosphorylation in chloroplasts

Protein phosphorylation has been recognized as an impor-tant regulatory principle in biochemistry for about half a cen-tury (reviewed in Cohen, 2002). The importance of reversible protein phosphorylation as a regulatory principle is further underpinned by the notion that at any given time about one-third of all cellular proteins are assumed to be phosphorylated

(Cohen, 2002). Moreover, protein kinases abound in eukar-yotic genomes, with more than 500 encoded in the human genome (Manning et al., 2002) and twice as many in Arabidopsis thaliana (Zulawski et al., 2014). While eukaryotes mainly possess serine/threonine (Ser/Thr) and tyrosine (Tyr) kinases, it was believed that bacteria recruit histidine kinases

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Abbreviations: CDPK, calcium-dependent protein kinase; CET, cyclic electron transport; ETC, electron transport chain; LHC, light-harvesting complex; NPQ, non-photochemical quenching; PC, plastocyanin, PET, photosynthetic electron transport; PQ, plastoquinone; PS, photosystem; ROS, reactive oxygen species.

Journal of Experimental Botany, Vol. 67, No. 13 pp. 3883–3896, 2016doi:10.1093/jxb/erw164 Advance Access publication 25 April 2016

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in combination with highly specific interacting response regulators to respond to external signals. However, in-depth genomic and high-throughput phospho-proteomics analyses revealed that eukaryote-like Ser/Thr and Tyr kinases are com-mon in bacteria (Kannan et al., 2007; Perez et al., 2008) and there is frequent phosphorylation of the respective residues in bacterial proteins (Macek et al., 2007; Pereira et al., 2011). The analysis of the evolutionary origins of Arabidopsis sig-nalling proteins revealed that a number of typical eukary-otic-like kinases, such as mitogen-activated protein kinases (MPKs) and a Ca2+-dependent kinase (CDPK), have been introduced by endosymbiosis. This provides evidence that these ‘typically eukaryotic signalling pathways’ have their evolutionary roots in the prokaryotic domain (Bayer et al., 2014) and is of particular relevance for chloroplasts as direct descendants of the primordial endosymbiont (Gould et al., 2008). Accordingly both evolutionarily ancient and ‘modern’ (i.e. eukaryotic type) kinases interact in the regulation of dif-ferent processes in chloroplasts (Bayer et al., 2012; Lundquist et al., 2012; Schönberg and Baginsky, 2012).

Phosphorylation in response to light conditions

A light-dependent phosphorylation of chloroplast thylakoid proteins was first described almost 40 years ago (Bennett, 1977), but it took another 25 years for the protein kinases responsi-ble to be identified in a genetic screen in Chlamydomonas rein-hardtii (Depege et al., 2003). The major kinases are the state transition kinases Stt7 and Stl1 in Chlamydomonas and their Arabidopsis orthologues, STN7 and STN8 (Bellafiore et al., 2005; Bonardi et al., 2005). Functional studies revealed that Stt7/STN7 is responsible for the phosphorylation of the major light-harvesting complex II (LHCII) while Stl1/STN8 phos-phorylates photosystem (PS) II core proteins (Bonardi et al., 2005). This finding led to the model that the phosphorylation of LHCII drives the movement of the LHCII antenna from PSII to PSI under changing light conditions, referred to as state transitions (Rochaix, 2014). When PSII is more excited (receives more energy) than PSI, LHCII is phosphorylated, detaches from PSII and binds to PSI in order to balance the energy distribution between the two photosystems (state 2). In turn, when PSI is over-excited, LHCII is dephosphoryl-ated by the protein phosphatase 1/thylakoid-associated phos-phatase 38 (PPH1/TAP38) phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and re-attaches to PSII (state 1). The molecular characterization of the STN7 kinase provided evi-dence for a redox regulation of its kinase activity (Lemeille et al., 2009), which is mediated by the PQ pool of the pho-tosynthetic electron transport chain. This is connected to the thioredoxin system and functions as the master regulator of various chloroplast processes (for a recent review see Balsera et al., 2014). In addition to the rather short-term regulation of light harvesting, chloroplast redox signals were also shown to regulate the expression of nuclear encoded plastid genes (Pfannschmidt et al., 1999; Bräutigam et al., 2009). This lat-ter process is called retrograde signalling and is discussed in

another article of this special issue (Kmiecik et al., 2016) and other recent reviews (see for example Chan et al., 2016).

Chloroplast phosphorylation in responses to different stresses

A number of recent studies have shown that different abiotic as well as biotic stresses have severe effects on plant growth affecting chloroplast functions. Photosynthesis is strongly impaired in response to abiotic stresses, such as heat, drought, cold, salinity, and high light stress. Photosynthesis also responds rapidly to pathogen infection (Göhre et al., 2012; de Torres Zabala et al., 2015; Gururani et al., 2015; Stael et al., 2015). Therefore, it is not surprising that not only the expres-sion of many nuclear encoded chloroplast proteins is altered in response to these stimuli (reviewed in Kmiecik et al., 2016), but also the chloroplast reacts by itself. A primary response is to control the generation of reactive oxygen species (ROS) resulting from photosynthesis (Foyer and Noctor, 2005). Therefore, the light-harvesting machinery can be modified to reduce the overall efficiency of light harvesting or to dissipate more energy as heat by non-photochemical quenching (NPQ) (Rochaix, 2014). Thereby, damage of the photosynthetic com-plexes by too much light (photoinhibition) is limited. This becomes particularly critical in combination with other stress factors (Barber and Andersson, 1992). Still, light-induced damage cannot be avoided entirely. Therefore, chloroplasts possess a repair machinery for the photosynthetic core pro-teins, most importantly the D1 protein of PSII (Edelman and Mattoo, 2008). The turnover of PSII core proteins is regulated by reversible protein phosphorylation (Koivuniemi et al., 1995), mediated by the state transition kinase STN8 (Bonardi et al., 2005) with clear differences between mono- and dicot plants (Nath et al., 2013). However, despite impli-cations for different stress conditions on photosynthesis, only a very few studies have looked at the role of thylakoid protein kinases under conditions other than different light/dark con-ditions (Pesaresi et al., 2011). Recently, Arabidopsis STN7 was found to be regulated by salinity or oxidative stress (Chen and Hoehenwarter, 2015), and drought stress increased the phosphorylation of the PSII core and D1 protein synthesis in pea (Giardi et al., 1996). Moreover, particularly in monocots, phosphorylation of thylakoid proteins and related NPQ have been suggested to be an acclimation strategy of chloroplasts to environmental stress (Chen et al., 2013; Marutani et al., 2014; Betterle et al., 2015).

Cross-talk of protein phosphorylation and Ca2+ signals

Calcium plays an important role in the chloroplast: Ca2+ is required for the water-splitting complex of PSII (Shen, 2015) and in the regulation of Calvin–Benson cycle enzymes (reviewed in Stael et al., 2012b). Moreover, Ca2+ signals act as an important secondary messenger in response to changing environmental conditions, including in the chloroplast (Dodd

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et al., 2010; Stael et al., 2012b). The regulation of photosyn-thesis by calcium signals was recently reviewed (Hochmal et al., 2015). Chloroplast Ca2+ signals were first shown in response to light–dark transitions (Sai and Johnson, 2002), and the chloroplast calcium sensing protein CAS was found to be phosphorylated by STN8 (Vainonen et al., 2008). Later CAS was also shown to regulate calcium signals in the plas-tid and strikingly also in the cytosol (Nomura et al., 2008; Weinl et al., 2008). CAS was found to be phosphorylated in a Ca2+-dependent manner (Stael et al., 2012a), and a recent study by Wang et al. (2016) indicated that CAS connects photosynthetic electron transport to stomatal closure. The authors used the electron transport inhibitor dibromothymo-quinone to trigger an over-reduction of the PQ pool leading to ROS-dependent stomatal closure. This required chloro-plastic H2O2 generation and was dependent on CAS func-tion and LHCII phosphorylation. While the exact molecular function of CAS is still unclear in higher plants, a role of CAS in light acclimation of photosynthesis was found in the green alga Chlamydomonas reinhardtii. CAS knockdown lines were impaired in NPQ due to a lack of the LHCSR3 pro-tein. The CAS mutant was also impaired in cyclic electron flow (CEF), and this inhibition could be rescued by increas-ing the extracellular Ca2+ concentration (Petroutsos et al., 2011; Terashima et al., 2012). CEF is also a mechanism for acclimation to adverse environmental conditions (Eberhard et al., 2008) leading to the proposal that CAS and Ca2+ (via CEF) could activate 1O2-mediated retrograde signalling to the nucleus (Terashima et al., 2012).

The current state of knowledge on thylakoid protein phosphorylation

Here we aim to provide a comprehensive survey of the current state of knowledge on phosphorylation of the main thylakoid complexes that perform the photosynthetic light reactions, excluding the auxiliary subunits of PSII. Therefore, we inte-grated the information about experimentally identified phos-phosites from three databases: PhosPhAt 4.0 (http://phosphat.uni-hohenheim.de) (Heazlewood et al., 2008; Durek et al., 2010), the Plant Protein Phosphorylation Database (P3DB, http://www.p3db.org) (Gao et al., 2009; Yao et al., 2014), and the database of Phosphosites in Plants (dbPPT, http://dbppt.biocuckoo.org) (Cheng et al., 2014). Additionally, we extracted phosphosites from 50 published studies (listed in Supplementary Data S1 at JXB online). The resulting set of phosphorylation sites that our analysis is based on is given in Supplementary Table S1. Unfortunately, mass spectrometry-based phosphoproteomics is prone to false positive identifica-tions. This issue is discussed in the meta-analysis of protein phosphorylation in Arabidopsis thaliana by van Wijk et al. (2014) and particularly for Tyr phosphorylation also by Lu et al. (2015a) as well as in two other papers of this special issue (Baginsky, 2016; Lohscheider et al., 2016). As a re-evaluation of spectra was beyond the scope of our study, we accepted all published phosphosites in the first round and subsequently filtered those in a similar way to van Wijk et al. (2014). First,

we excluded putatively phosphorylated peptides lacking an unambiguous assignment of the exact phosphorylation site. In a second step we assigned the remaining data to two groups of differing confidence. Phosphosites that were consistently identified in two studies or more were placed in the ‘high confidence’ group and are specifically indicated in Figs 2–3. Of course, this will not remove all false positive identifica-tions and also does not imply that all phosphosites that have been identified only once present false positives per se. The resulting dataset (Supplementary Table S1) provides evidence for 426 unambiguous phosphorylation sites in 13 species. Of these only 85 (20%) belong to the high confidence group. Of the total phosphosites, 54.7% have been found in dicots, 6.6% in monocots, and 30% in the green alga Chlamydomonas rein-hardtii. Surprisingly, the number of common phosphosites among these three groups is rather low: 14 sites between dicots and monocots, nine between dicots and green algae, and none between monocots and green algae (Fig. 1). The three groups show only three common phosphosites, which all belong to the PSII core: Thr-2 of protein D1, Thr-2 of D2 and Thr-15 of CP43. This low overlap between the studies could be due to several factors. Firstly, there is a huge dif-ference in the number of studies. While 23 studies have been performed in Arabidopsis, only a few have been performed in other higher plants (for example two in rice, three in maize, and three in wheat and barley). Most of the algal data come from only one study (Wang et al., 2014). Secondly, there is a significant difference in the organization of the light-har-vesting machinery between algae and higher plants (Rochaix, 2014). This is particularly important as LHC proteins consti-tute the largest group of phosphorylated thylakoid proteins. Finally, sites that are phosphorylated in one species but not in another might also point towards different modes of regu-lation that have changed during evolution. For example the D1 core protein of PSII is encoded by a single gene in higher

Fig. 1. Venn diagram depicting the distribution of experimentally determined phosphosites among green algae, monocots and dicots. For a full list of phosphosites see Supplementary Table S1.

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plants, and D1 is modified by phosphorylation. In contrast, D1 is encoded by a small gene family in cyanobacteria, which slightly differ at the amino acid level, and these genes are dif-ferentially expressed in response to environmental stimuli (Edelman and Mattoo, 2008).

We have summarized the data using Arabidopsis as the model for higher plants and Chlamydomonas represent-ing the green algae, as most data are available for those two species (Fig. 2). We used the nomenclature of photo-synthetic complexes as listed in the reviews by Allen et al. (2011) and Rochaix (2014) for the light-harvesting com-ponents. In summary, all major complexes are subject to phosphorylation in both species, but different sites might be used. In this context, we need to emphasize that most of these phosphosites were detected in studies without the application of a particular stress or environmental change related to photosynthesis. Some studies were performed under different light conditions (i.e. light or dark), and only a very few studies were performed applying other

specific stress conditions such as drought, cold, and salt stress. Moreover, quite a number of studies used plant tis-sue derived from cell cultures. All these aspects may result in significant metabolic and physiological differences (van Wijk et al., 2014).

Evolutionary conservation of phosphorylation sites – hints for further functional studies

While functional implications of protein phosphorylation are quite well understood for PSII and the related light-harvesting complexes, a big gap in our knowledge exists for the functional consequences of phosphorylation at the other complexes. Nevertheless, quite a number of phospho-sites have been identified there too (Fig. 2). So how could a functional link be established, and where to start with fur-ther experiments? To provide some ideas in this direction, we

Fig. 2. Distribution of phosphosites among thylakoid proteins. Overview of identified phosphorylation sites in the subunits of the main components of light reactions in Arabidopsis thaliana and Chlamydomonas reinhardtii. Red circles represent phosphosites that were reported in only one publication, while brown squares represent phosphosites reported in more than one publication or isoform (a full list of phosphosites with respective references is provided in Supplementary Table S1). The number in every circle or square indicates the phosphosite position in the full protein sequence (initial translation product). Only unambiguous phosphosites are included in the analysis. The position of circles and squares is not indicative of the precise position of phosphosites. Shape and localization of subunits do not exactly resemble the actual structure. In the case of proteins having more than one isoform, only the isoform for which more experimental data have been reported is shown. Subunits encoded by the plastid genome are coloured blue; nuclear-encoded subunits are coloured yellow. Cyt b6f: cytochrome b6f; Fd: ferredoxin; FNR: ferredoxin–NADP+ oxidoreductase; LHCI: light harvesting complex I; LHCII: light harvesting complex II; LHCSR: stress-related chlorophyll a/b binding protein; PC: plastocyanin; PSI: photosystem I; PSII: photosystem II. Every subunit is identified by its codifying gene, whose name is given by the letter or number depicted on the subunit, preceded by a prefix that refers to the protein complex: atp for ATP synthase, lhca for LHCI, lhcb for LHCII, lhcsr for LHCSR, pet for Cyt b6f, psa for PSI, psb for PSII.




considered the evolutionary conservation of phosphorylation sites in these proteins.

The rationale behind this is that for a highly conserved process such as photosynthesis in higher plants, it should be expected that regulatory mechanisms are conserved as well. A regulation by phosphorylation requires the presence of a ‘phosphorylatable’ amino acid (i.e. Ser, Thr, or Tyr) at a par-ticular position in a target protein, suggesting that the con-servation of such a residue over time indicates a functional role of this phosphorylation. Functionally, a phosphoryla-tion adds a negative charge to amino acid side chains, and sometimes negatively charged amino acids (i.e. Asp/Glu) can mimic the phosphorylated state of a protein. In a compara-tive genomics approach, Ferrell and colleagues showed that indeed phosphorylatable amino acids evolved from Asp/Glu residues (Pearlman et al., 2011). One illustrative exam-ple in support of this hypothesis is the Calvin–Benson cycle enzyme transketolase, which is phosphorylated in a Ca2+-dependent manner at a Ser residue. This Ser is homologous to a conserved Asp residue present in the transketolase of mosses, green algae and cyanobacteria (Rocha et al., 2014). Systematic studies to find a general link between the evolu-tionary conservation of a phosphorylation site and its regu-latory effect across entire phosphoproteomes indicated some additional important aspects: phosphosites are, on average, more conserved than their non-phosphorylatable equivalent residues when they are localized in disordered regions of the protein, and more importantly, they are much more conserved if they have a characterized function (Landry et al., 2009). This again highlights the advantage of using evolutionary information to identify functional regulatory phosphoryla-tion sites (Landry et al., 2009). Moreover, the conservation of the phosphorylation state is a better indicator for functional relevance than sequence conservation of the phosphorylation site (Beltrao et al., 2012). In fact a recent large-scale analysis of the evolutionary histories of phosphorylation motifs in the human genome revealed that highly conserved phospho-motifs are likely to be involved in similar signalling networks with functionally important roles (Yoshizaki and Okuda, 2015).

To study the evolutionary conservation of phosphoryla-tion sites in thylakoid proteins we performed an orthologue search for all phosphoproteins included in this review. We downloaded the protein sequences for 58 photosynthetic phosphoproteins (86 sequences if we count all isoforms) from the TAIR 10 database (http://www.arabidopsis.org; last accessed 02/02/2016) (Berardini et al., 2015; Lamesch et al., 2012) and performed for each of these proteins a tar-geted orthologue search to determine its presence–absence pattern in 41 photosynthesizing species, 36 downloaded from Phytozome v10 (https://phytozome.jgi.doe.gov; last accessed 02/02/2016) supplemented with proteomes of five species downloaded from Uniprot (http://www.uniprot.org). These were Brassica napus, Solanum oleracae, Hordeum vul-gare, Triticum aestivum, and Synechocystis sp. PCC 6803. The list of analysed species is given in Supplementary Table S2 and covers 29 dicots, eight monocots, Selaginella moellendorffii for Lycopodiophyta, Physcomitrella patens

for mosses, Chlamydomonas reinhardtii for green algae, and Synechocystis for cyanobacteria. In these species we used each protein as a seed for a targeted orthologue search with HaMStR-OneSeq (Ebersberger et al., 2014), an extension of the profile hidden Markov model (pHMM)-based ortho-logue search tool HaMStR (Ebersberger et al., 2009; http://sourceforge.net/project/hamstr). The tool was run with the following parameter settings: ‘-strict’, ‘-coreStrict’, ‘-check-CoorthologsRef’, ‘-coreCheckCoorthologsRef’ and ‘-rep’. Subsequently, we aligned the sequences in each of the result-ing orthologous groups with MAFFT v7.266 (Katoh and Standley, 2013).

In the next step, we generated protein alignments of all proteins in the most important photosynthetic complexes. An overview of this analysis is shown in Fig. 3. It includes a subset of 22 proteins that are representative of the main components of light reactions. The position of the identi-fied phosphosites as well as the level of conservation at these sites is indicated in a schematic presentation of the protein structure (Fig. 3). In this subset 142 phosphosites were found. Thirty-eight phosphosites are highly conserved during evolu-tion, i.e. they have been found in all species analysed (listed in Supplementary Table S2), and 12 of these 38 are reported in more than one publication. Twenty-nine sites were found phosphorylated in dicots but are not conserved as Ser, Thr or Tyr in monocots. Instead, only one site was found phos-phorylated in monocots, but not conserved in dicots, i.e. Thr-489 in chloroplast ATP synthase β-subunit (del Riego et al., 2006). This could be a bias due to a much lower amount of experimental data available in monocots compared with dicots. We will now discuss the individual complexes start-ing with the light-harvesting complex and PSII where most information is available and try to relate this knowledge to a functional role of phosphorylation.

Photosystem II and light harvesting complex II

Photosystem II (PSII) is a dimeric complex that performs water-splitting at the onset of photosynthetic light reactions to fuel the electron transfer chain (Nelson and Junge, 2015; Shen, 2015). Most of the required energy for this process is collected by the light-harvesting complexes composed of pig-ment–protein complexes, called ‘antennae’. They absorb the light and transfer its energy to the PSII core, composed of D1, D2, CP43 and CP47 proteins. In land plants every PSII mon-omer binds three monomeric antennae, called CP24, CP26 and CP29, while Chlamydomonas lacks CP24 (Merchant et al., 2007). In addition, the light-harvesting complex II (LHCII) constitutes a trimeric antenna, composed of Lhcb1, Lhcb2 and Lhcb3 proteins, in various combinations, where every PSII dimer can bind a variable number of LHCII trim-ers (Nelson and Junge, 2015; Rochaix, 2014).

Lhcb1 and Lhcb2 are the main components of the LHCII trimers and both are phosphorylated, but neither alone is sufficient to drive state transitions. Lhcb1 and Lhcb2 have different and complementary functions in light harvesting



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Fig. 3. Evolutionary conservation of potential phosphosites. Here we selected 22 proteins from all major complexes involved in photosynthetic light reactions. Based on protein sequence alignment, the conservation of the serine, threonine and tyrosine residues that were found phosphorylated in one or more species was analysed in the cyanobacterium Synechocystis sp. PCC6803, in the green alga Chlamydomonas reinhardtii, in the moss Physcomitrella patens, in the spike moss Selaginella moellendorffii, and in several monocot and dicot species (see Supplementary Table S2 for complete list of species). Every pin symbol indicates a phosphosite that was experimentally found in at least one species. The numbers above pins indicate the positions of phosphosites in the Arabidopsis thaliana protein sequence or, if the phosphosite was found in a different species, the corresponding position in the Arabidopsis sequence. Red squares around numbers indicate the phosphosites that are reported in more than one publication or in different protein isoforms. Circles indicate the presence of serine, threonine, or tyrosine in the corresponding phosphosite position. In monocots and dicots, a red circle indicates that serine, threonine or tyrosine was present in all the species in which an orthologue was found, a yellow circle indicates their presence in the range of 80–99% of the species, and a light blue circle indicates their presence in less than 80% of species. The black border around some circles indicates in which species or taxonomic group the phosphosite was experimentally found. Only unambiguous phosphosites present in highly conserved protein domains are included in the analysis. A question mark indicates lack of sequence information. For antenna proteins, Synechocystis and Chlamydomonas were not included. The gene encoding the protein is indicated in parentheses, after the protein name. PP: pro-peptide; TM: trans-membrane domain; TP: transit peptide.




and are phosphorylated to a different extent in the com-plexes bound to PSII or PSI (Longoni et al., 2015). Most of the phosphorylated forms of Lhcb1 and Lhcb2 are present in super- and mega-complexes that comprise both PSI and PSII (Leoni et al., 2013). PSI–LHCII complexes were found to be highly enriched in the phosphorylated forms of Lhcb2 (Leoni et al., 2013; Crepin and Caffarri, 2015; Longoni et al., 2015) and in the absence of Lhcb2, the PSI–LHCII complex was not formed (Pietrzykowska et al., 2014). The phospho-rylation of Ser-30, Thr-38, Ser-48 and Ser-53 in Lhcb1 and Thr-40 in Lhcb2 has been reported in several publications (Supplementary Table S1). Thr-38 and Ser-48 in Lhcb1 and Thr-40 in Lhcb2 are highly conserved during evolution, while Ser-53 in Lhcb1 is conserved only in angiosperms, and Ser-30 (experimentally observed in Lhcb1 and Lhcb2) is conserved only partially in dicots (Fig. 3).

Nevertheless, the traditional ‘state transition’ model cannot explain all experimental observations. LHCII phosphorylation changes also upon different light intensities (Rintamäki et al., 1997; Rintamäki et al., 2000) and occurs not only transiently during short-term acclimation but also under constant white light (Tikkanen et al., 2006; Grieco et al., 2012; Wientjes et al., 2013b). This ensures that a part of LHCII, contrary to the ter-minology, is also a stable antenna of PSI (Galka et al., 2012). Steady-state LHCII phosphorylation, in cooperation with NPQ, is crucial for regulation of electron transfer and pro-tection of PSI upon fast fluctuations of light intensity under conditions that resemble environmental light changes such as leaf shading or cloud movement (Grieco et al., 2012). In both steady-state illumination and short-term change of light inten-sity, also the fraction of LHCII that remains tightly bound to PSII is phosphorylated (Grieco et al., 2012; Wientjes et al., 2013a). Plants and green algae possess also LHCII trimers that are not tightly bound to PSII or to PSI, and have been defined as ‘extra LHCII’ (Peter and Thornber, 1991;Broess et al., 2008; van Oort et al., 2010; Drop et al., 2014a). The physiological role of extra LHCII has long been neglected although it was estimated that it accounts for about half of the total LHCII. It was proposed that it forms an LHCII ‘lake’, i.e. a network of LHCII trimers that can transfer energy to each other and to both PSII and PSI (Grieco et al., 2015). In this model the phos-phorylation of LHCII is not necessary for the energy trans-fer from LHCII to PSI, but rather it contributes to regulating the distribution of absorbed light energy between PSII and PSI. Recent studies confirmed the energy transfer from extra LHCII to PSI, even in the absence of LHCII phosphorylation (Benson et al., 2015; Akhtar et al., 2016).

Similarly to LHCII, PSII is steadily phosphorylated under continuous white light illumination (Tikkanen et al., 2006; Grieco et al., 2012; Wientjes et al., 2013b). The dynamics of PSII phosphorylation is coordinated with changes of LHCII phosphorylation. In state transition experiments where the light quality is changed, PSII and LHCII are concomitantly phosphorylated and dephosphorylated, while upon modifi-cation of light intensity PSII and LHCII show an opposite trend of phosphorylation (Tikkanen et al., 2010). Upon expo-sure to high light the D1 subunit of PSII is damaged and the increase of PSII core phosphorylation is important for its

repair, facilitating its migration from grana to stroma lamel-lae, where damaged D1 is replaced (Koivuniemi et al., 1995; Tikkanen et al., 2008). The effects of PSII phosphorylation are PSII monomerization (Aro et al., 2005), grana de-stacking (Fristedt et al., 2009; Herbstova et al., 2012), and increased membrane fluidity (Goral et al., 2010; Herbstova et al., 2012). All modifications accelerate protein migration in the thylakoid system. However, a conformational change induced by phos-phorylation cannot be excluded. PSII de-phosphorylation is then required for efficient D1 degradation in stroma lamellae (Koivuniemi et al., 1995; Rintamäki et al., 1996).

Differences in light acclimation between higher plants and algae

State transitions in land plants show some differences com-pared with green algae. CP29 and CP26 have been shown to participate in antenna relocation only in Chlamydomonas (Takahashi et al., 2006; Iwai et al., 2008). The portion of LHCII that is relocated during state transitions was estimated to be 80% for Chlamydomonas and only 15% for higher plants (Allen, 1992; Delosme et al., 1996). In Chlamydomonas, state transitions induce a switch from linear to cyclic elec-tron flow (Finazzi et al., 2002). This would constitute an important functional difference between green algae and plants in coping with stress. While state transitions func-tion as a mechanism to fine-tune linear electron flow in land plants, state transitions induce a shift from oxygenic photo-synthesis (producing both NADPH and ATP) to a bacterial type photosynthesis (producing only ATP) in green algae. The coupled ATP accumulation would give algae an advan-tage for re-acclimation when the stress terminates (Finazzi and Forti, 2004). In Chlamydomonas only a small fraction of LHCII moves from PSII to PSI during state transitions, while the rest of LHCII, detached from PSII, plays a role in energy dissipation, thus protecting PSII from excessive dam-age (Unlu et al., 2014). Furthermore, in Chlamydomonas state transitions are induced differently compared with plants: state 1 can be induced by inhibiting PSII with DCMU, thus provoking the oxidation of the PQ pool, and reversed in anaerobic conditions in the dark, which induces PQ reduc-tion. Notably, darkness induces LHCII de-phosphorylation in both algae and plants. In Chlamydomonas, NPQ is medi-ated by LHCSR3, a member of the light-harvesting complex stress-related (LHCSR) protein family (Petroutsos et al., 2011; Allorent et al., 2013). Expression of LHCSR3 and state tran-sitions cooperate to deal with high light stress. Phosphosites in LHCSR2 and LHCSR3 proteins were reported by Wang et al. (2014). Importantly, LHCSR proteins are present in algae and the moss Physcomitrella patens, but not in vascular plants, where PsbS has a similar function (Gerotto et al., 2015). In Chlamydomonas only LHCII type I, II, and IV are phospho-rylated when they are associated with PSI, while LHCII type III and CP29 are not. In contrast, CP29 is phosphorylated when it is associated with PSII (Drop et al., 2014b). LhcbM2/7 and LhcbM5 are involved in state transitions, while LhcbM1 is important for NPQ (Takahashi et al., 2006).




CP29 has different phosphorylation sites in dicots and monocots

CP29 is one of the monomeric antennae of PSII and posi-tioned between PSII and the LHCII trimers. It was proposed that CP29 phosphorylation leads to disassembly of PSII–LHCII complexes and functions to down-regulate photo-synthetic activity under stress conditions in monocots (Chen et al., 2009). In rice the STN7-dependent CP29 phospho-rylation contributes to NPQ under high light stress (Betterle et al., 2015). In dicots CP29 is phosphorylated only under high light stress (Hansson and Vener, 2003; Tikkanen et al., 2006; Chen et al., 2009; Fristedt and Vener, 2011), while in monocots CP29 was found to be phosphorylated in many dif-ferent stress conditions (Bergantino et al., 1995; Bergantino et al., 1998; Pursiheimo et al., 1998; Chen et al., 2009; Liu et al., 2009). For example, drought stress induced CP29 phos-phorylation with concomitant decrease of PSII phosphoryla-tion in barley (Liu et al., 2009).

In Arabidopsis Thr-37 of CP29 is phosphorylated (Hansson and Vener, 2003; Reiland et al., 2009; Fristedt and Vener, 2011; Yang et al., 2013), but this Thr is conserved only in some dicots (Fig. 3). Bonhomme et al. (2012) reported a concomitant increase of CP29 and PsbH phosphorylation in maize upon application of drought stress, but no data on photosynthetic activity were presented in that study. Interestingly, CP29 and PsbH are placed in close proximity in the PSII–LHCII super-complex structure, suggesting possible functional implica-tions for their phosphorylation. Finally, CP24, another minor antenna of PSII, is reversibly phosphorylated upon changes of light intensity in Lycopodiophyta (Ferroni et al., 2014).

Proteins stabilizing the oxygen evolving complex

Photosynthetic water-splitting occurs in the Mn4CaO5 clus-ter at PSII. The stabilization and optimal activity of the clus-ter require the PsbO, PsbP, PsbQ, and PsbR subunits of PSII in green algae and plants (Shen, 2015). Many phosphosites were found in these proteins in Chlamydomonas as well as in plants (Fig. 2). An interesting case is PsbQ-2, whose Ser-125 was found phosphorylated in Chlamydomonas, Selaginella, and Arabidopsis. This residue is not highly conserved in dicots, while it is present in Physcomitrella patens but not in mono-cots (Fig. 3). In the same protein, Thr-127 phosphorylation was observed in two independent studies. However, this residue is conserved only partially in dicots. The role of PsbO, PsbP, PsbQ and PsbR phosphorylation is still unknown. Another intrigu-ing question emerging from this finding is which kinase cataly-ses their phosphorylation in the lumen. Still, we do not know how many of those sites present false positive identifications and beyond that most phosphosites were identified only once.

The cytochrome b6f complex

The cytochrome b6f complex (Cyt b6f) is a central component of the photosynthetic electron transport chain (ETC) that

mediates the electron transfer from PSII to PSI accompanied by proton pumping into the thylakoid lumen. Cyt b6f reduces plastocyanin (PC) and oxidizes plastoquinol (PQH2), which is the rate-limiting step in the inter-system chain of the ETC (Tikhonov, 2014). In addition to the mechanisms discussed before in the context of light harvesting, photosynthetic elec-tron flux needs to be adjusted to metabolic demand, which is called ‘photosynthetic control’ and mediated via the phos-phate potential (Tikhonov, 2014). Cyt b6f and chloroplast ATP synthase are the major regulators in this process (Tikhonov, 2014; Schöttler et al., 2015), which makes them also potential targets for regulation. Cyt b6f plays an important role in state transitions. The Stt7/STN7 kinase is activated upon binding of plastoquinol to the Qo site of Cyt b6f (Zito et al., 1999), and its N-terminal domain, required for its activity, interacts directly with the Rieske protein of the Cyt b6f (Shapiguzov et al., 2016). At first sight, two remarkable differences in the distribution of phosphosites in Cyt b6f become evident between Chlamydomonas and Arabidopsis: firstly, PC was found to be phosphorylated in Chlamydomonas but not in Arabidopsis, and secondly, the Cyt b6f of Chlamydomonas includes a phosphorylated subunit called subunit V (or PetO), which is absent in land plants. Subunit V is phospho-rylated at Thr-161 and Thr-178, located in the C-terminal, stroma-exposed domain (Hamel et al., 2000). This is par-ticularly interesting because subunit V is a transmembrane protein with two hydrophilic domains exposed to both sides of the thylakoid membrane. Thereby it could function as a signal transducer, thus subtending important functional dif-ferences between plants and Chlamydomonas. It has recently been shown that phosphorylation of subunit V is depend-ent on STT7 kinase and is higher in anoxia than in the high light condition, but it is not required for activation of CEF (Bergner et al., 2015). Nevertheless, subunit V protein con-tributes to the stimulation of CEF in anoxia, interacting with other effectors of CEF (Takahashi et al., 2016).

Photosystem I and light harvesting complex I

Photosystem I (PSI) oxidizes PC and reduces ferredoxin (Fd) at the end of the photosynthetic electron transport chain (Jensen et al., 2007; Nelson and Junge, 2015). Therefore, from the functional point of view, it is placed at the inter-face between light reactions and stroma metabolism. To our knowledge, no studies on the physiological role of PSI phos-phorylation have been published. Phosphosites in PSI–LHCI were mostly detected in non-stressed plants, and therefore it is difficult to draw conclusions on the function of its phospho-rylation. Surprisingly, our analysis revealed that several sites that were found phosphorylated in PSI and LHCI proteins in Arabidopsis were conserved only in some dicots (Fig. 3). For instance Ser-35 of Lhca4, with a reported phosphorylation in five studies, is conserved only partially in dicots, as well as Ser-38 and Ser-68 (Fig. 3). Both PsaF and PsaN, which are involved in binding of PC, are phosphorylated (Figs 2 and 3). Interestingly, the phosphorylation of PsaN protein was found



to be dependent on Ca2+ (Stael et al., 2012a), thus indicat-ing a crosstalk between calcium signalling and protein phos-phorylation. Phosphosites are reported also on PsaD and PsaE subunits, which are important for binding of Fd and Fd–NADP oxidoreductase (FNR). PsaD is phosphorylated at Thr-48 in a light- and redox-dependent manner (Hansson and Vener, 2003), and FNR was found phosphorylated as well (Fig. 3).

Phosphosites have also been reported at some of the PSI subunits involved in docking of LHCII, i.e. PsaH, PsaL, and PsaK (Jensen et al., 2007). In particular, five phosphosites were determined in PsaL, which is required for the stabiliza-tion of PsaH and PsaO (Jensen et al., 2007). One of these sites (Ser-46), discovered in two studies in Arabidopsis, is partially conserved in dicots and in Chamydomonas, but is absent in monocots and Physcomitrella (Fig. 3). PSI phos-phosites could therefore have important physiological impli-cations, since they can potentially be involved in regulating the binding of PC, Fd and LHCII to PSI. In this context it would be interesting to see whether the phosphorylation of PC in Chlamydomonas plays a role in regulating the interac-tion of PC with PSI, or with the Cyt b6f complex. Seven phos-phosites were detected for PC in Chlamydomonas (Fig. 2). Phospho-Ser-57, in particular, was reported in three differ-ent studies and the residue is conserved also in Synechocystis, but it is absent in all plant species included in our analysis (Fig. 3). So far, no functional studies have been carried out on this potential difference in the regulation of the ETC between algae and land plants. The first obvious question is whether PC phosphorylation is a specific requirement for acclimation of photosynthesis in an aquatic environment. Moreover, is PC phosphorylation somehow functionally related to the phosphorylation of PsaF and PsaN subunits of PSI in green algae?

ATP synthase

The chloroplast ATP synthase couples the electrochemical trans-thylakoid proton gradient generated by light reactions to the production of ATP in the chloroplast stroma (Junge and Nelson, 2015). Many phosphosites have been identified in chloroplast ATP synthase in different species, and for some of them also physiological functions have been suggested (Fig. 2). However, there is still only a small overlap of identi-fied phosphosites between different species (Fig. 3). Therefore the question is whether this is due to false positives or techni-cal issues. Or, instead, does it reflect biological differences, for example different strategies to regulate ATP synthase activity in different species?

Two studies (Reiland et al., 2011; Roitinger et al., 2015) reported two common phosphosites in the Arabidopsis ATP synthase α-subunit (AtpA gene): Ser-139 and Ser-488 (Figs. 2–3). Ser-139 is conserved in all land plants included in our analysis, while the latter is present as serine or threonine only in some angiosperms (Fig. 3). To our knowledge, there are no functional studies on these two phosphosites, but it is tempt-ing to suggest that the phosphorylation of Ser-139 may be

related to acclimation to land environments, while phospho-Ser-488 could be unrelated to essential functions. Notably, Ser-139 is conserved in all the plant species we analysed, while the corresponding position in Chlamydomonas is occupied by an alanine, but it is replaced by a negatively charged amino acid (Glu) in Synechocystis.

Chloroplast ATP synthase can be deactivated/activated by oxidation/reduction of a disulfide bridge present in the γ-subunit. This regulative mechanism is required for switch-ing off/on the ATP synthase upon dark/light transition (Junge and Nelson, 2015). Kohzuma et al. (2013) suggested that the redox regulation of the γ-subunit is not involved in acclimation to environmental stresses, based on the observa-tion that an Arabidopsis mutant with altered redox potential of the γ-subunit disulfide–sulfhydryl couple showed a normal response to varying CO2 concentration. The response of chlo-roplast ATP synthase to environmental stress could in part rely on the phosphorylation of the β-subunit, which includes several phosphosites (Fig. 3 and Supplementary Table S1). Ser-8 and Ser-13, which were found phosphorylated at the end of the night (Reiland et al., 2009), are possibly involved in the inactivation of ATP synthase in the dark. Interestingly, Ser-8 and Ser-13 residues were found conserved only in angi-osperms (Fig. 3). Six Ser/Thr residues were found phospho-rylated in the β-subunit of the thylakoid ATP synthase in barley, including the conserved Thr-179 that is also phospho-rylated in the β-subunit of human mitochondria (del Riego et al., 2006). Some of these phosphorylation events were pro-posed to be involved in the regulation of the ATP synthase by phosphorylation and 14-3-3 proteins. Bunney et al. (2001) found that 14-3-3 proteins function as regulators of the mito-chondrial and chloroplast ATP synthase. 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate multiple enzymes in plants, animals, and yeast. In that work, 14-3-3 proteins were found associ-ated with the ATP synthase F1 β-subunit in a phosphoryl-ation-dependent manner. Addition of recombinant 14-3-3 proteins did strongly reduce the activity of the ATP synthases in both organelles and the down-regulation of chloroplast ATPase activity in darkness was prevented by a phosphopep-tide containing the 14-3-3 interaction motif. From these data the authors concluded that regulation of the ATP synthase by phosphorylation and subsequent 14-3-3 binding repre-sents a mechanism for plant acclimation to environmental changes such as light–dark transitions, anoxia in roots, or fluctuations in nutrient supply. It has been shown before that the chloroplast β-subunit is phosphorylated by chloroplast casein kinase II (Kanekatsu et al., 1998), although it is not yet clear whether the putative 14-3-3 recognition motif, which is conserved in the C-terminal domain of both chloroplast and mitochondrial ATP synthases, is involved. Strikingly, this putative 14-3-3 binding motif lies in close proximity to the so-called DELSEED sequence (Abrahams et al., 1994), which contains a casein kinase II phosphorylation motif (Kanekatsu et al., 1995).

Ser-135 of the β-subunit was found to be phosphorylated in Arabidopsis and in Chlamydomonas, but functional implica-tions were not tested. Our analysis suggests that it is conserved




from green algae to angiosperms, although not in all dicot species. Several studies confirm the phosphorylation of the γ-subunit at Ser-243 and Ser-347 in Arabidopsis, and Ser-347 was reported to be phosphorylated also in Chlamydomonas. Although both sites are highly conserved, a physiological role is not known. Intriguingly, Ser-243 is positioned six residues upstream of the disulfide bridge that is responsible for the redox regulation of ATP synthase activity (discussed above). Seven phosphosites were found in subunit II (AtpG gene) and only one of them (Ser-209) is highly conserved in all species. It is surprising that all the other sites are not found in mono-cots, but show some conservation in the other species.

Schmidt et al. (2013) identified 10 phosphosites in the spin-ach chloroplast ATP synthase, at subunits α, β, δ, ε and II, and suggested a role for phosphorylation in regulating nucle-otide binding and stability of the chloroplast ATPase. Among these sites, however, only Ser-8 at the β-subunit was found phosphorylated in another species (Arabidopsis thaliana). In our analysis, this site is highly conserved in dicots and mono-cots, but is absent in all other species, which could suggest a physiological role specific to angiosperms. In general, these findings fit well with observed differences in substrate specifi-cities between the chloroplast casein kinase II (pCKII) from Arabidopsis and rice (Lu et al., 2015b). pCKII is one of the major kinase activities in chloroplasts (Reiland et al., 2009; Bayer et al., 2012) and could be one of the candidate kinases for ATP synthase as discussed before.

Conclusions

Since the discovery of protein phosphorylation as an impor-tant means of regulation of photosynthesis in the end of the 1970s, we have seen a rapid increase in the number of identified phosphorylation sites in thylakoid proteins due to technical improvements in mass spectrometry-based phosphoproteom-ics. However, this has not equally increased our understand-ing of the biological meaning of these events. A functional characterization of identified phosphosites is technically very demanding and time- as well as labour-intensive. Therefore strategies are required to indicate which ones of those various sites seem most promising for further study. Our survey shows that multiple phosphorylation hotspots emerge from com-parative analysis, which could indicate a functional relevance. This is particularly interesting for phosphorylation events at other complexes than PSII and LHCII, because there the effects of phosphorylation on the regulation of light-harvest-ing and stability of PSII core proteins are well understood. In contrast, at the Cyt b6f complex, PSI and the ATP synthase, functional roles of phosphorylation are not yet known. In this context, it is remarkable that almost all the phosphosites we found at those complexes were identified in studies that were not specifically targeting photosynthesis. Thus it will be of the utmost interest to investigate the role of these phosphosites in the context of acclimation of photosynthesis to changing environmental conditions. A useful strategy that could be employed here to further narrow down the number of tar-get sites to study in detail is to consider their evolutionary

conservation. Using this approach, we found highly conserved sites carrying a phosphorylatable amino acid at the respective position in all species, which would point towards an essen-tial function. In addition we also found interesting differences, where a phosphorylatable amino acid was highly conserved except for monocots. In line with such differences, different substrate specificities or regulatory functions were reported for the pCKII as well as STN8 between Arabidopsis and rice (Nath et al., 2013; Lu et al., 2015b). Accordingly, it would be highly interesting to see if these could reflect different strate-gies, for example, to cope with different environmental con-ditions. Finally, it also became clear that many phosphosites are present at the luminal side and some of them are highly conserved. This makes them prime candidates for future study and leads to the question of which kinase is responsible for their phosphorylation. However, it has also to be considered that a number of the ‘novel’ phosphosites might represent false positive observations. Therefore, only further experimen-tal studies will clarify this point.

Supplementary data

Supplementary data are available at JXB online.Data S1: References. List of references for published

phosphosites.Table S1. List of phosphosites reported in Synechocystis,

Chlamydomonas reinhardtii and land plants. Table S2. Complete list of species for orthologue search.

AcknowledgementsThis work was supported by the EU Marie Curie Initial Training Network (ITN) CALIPSO (GA ITN-2013 607 607) and Austrian Science Fund (FWF) with project P-25359 to M.T. We thank Francy El Souki for support in preparing the figures and Ella Nukarinen and Bernhard Wurzinger for critical discussions. Finally we apologize to all authors whose work could not be discussed here due to space limitations.

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