MINI-REVIEW Plant Signaling & Behavior 10:12, e1058462; December 2015; © 2015 Taylor and Francis Group, LLC
Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii Huidan Xuey, Sonja Verena Bergnery, Martin Scholz, and Michael Hippler* Institute of Plant Biology and Biotechnology; University of M€ unster; M€ unster, Germany y
These authors equally contributed to this work.
Keywords: Chlamydomonas reinhardtii, excess light to heat dissipation, light-harvesting, LHCSR3, photosynthesis
Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photooxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii.
LHCSR3 is required for qE in algae and mosses Photosynthetic organisms convert solar energy into chemical energy by employing a series of reactions in and at the thylakoid membrane resulting in light-dependent water oxidation, NADP reduction and ATP formation.1 Hereby light is captured by the intrinsic chlorophyll a antenna systems of photosystem I (PSI) and photosystem II (PSII) as well as via associated chlorophyll a/b binding light harvesting complexes (LHC). The LHC superfamily is a monophyletic group and includes high light-induced proteins (HLIPs), stress-enhanced proteins (SEPs), PSBS and early light-inducible protein (ELIP).2,3 It is well accepted that LHC proteins not only functionally collect light energy for photosynthesis but also protect plants and algae from excess light. In such conditions, specialized light-harvesting proteins facilitate de-excitation of excess photons to avoid photodamage in high light. The fastest response to excess light is provided by a mechanism called non-photochemical quenching (NPQ). An important constituent of NPQ is the energy-dependent quenching (qE), which regulates the thermal dissipation of excess absorbed light energy. To become functional it requires the acidification of the thylakoid lumen via photosynthetic electron transfer. Thus the qE mechanism is functionally coupled to photosynthetic electron transfer and provides so efficient photo-protection. In *Correspondence to: Michael Hippler; Email: [email protected] Submitted: 05/26/2015; Accepted: 06/01/2015 http://dx.doi.org/10.1080/15592324.2015.1058462
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vascular plants, PSBS, a protein of the LHC protein superfamily having 4 transmembrane helices, is essential for efficient qE.4 PSBS functions as a lumenal pH sensor and regulates qE in vascular plants via protonation of 2 glutamate residues (Glu-122 and Glu-226), which both can be modified with dicyclohexylcarbodiimide (DCCD).5,6 Moreover, in vascular plants, acidification of the lumen activates the biochemical conversion of the light-harvesting violaxanthin to antheraxanthin and zeaxanthin via vioaxanthin de-epoxidase, that is required for effective qE.7,8 In green algae, the qE capacity is dependent on LHCSR3.9 Similar to vascular plants, also in green algae low lumenal pH is necessary for qE formation,10 which likely operates via protonation of acidic groups in LHCSR3, as LHCSR3 binds DCCD when associated to PSII-LHCII supercomplexes.11 LHCSR-like proteins (formerly named LI818-like or LHCX-like proteins) were originally identified as light-induced mRNA transcripts and are present in green algae, haptophytes, heterokonts and chromerida,12 but absent in vascular plants and red algae.2 There are 3 nuclear encoding lhcsr genes in Chlamydomonas reinhardtii, which are lhcsr1, lhcsr3.1 and lhcsr3.2.9 Both lhcsr3.1 and lhcsr3.2 encode an identical 259-amino-acid polypeptide. The other, paralogous gene lhcsr1 codes for a protein of 253 residues with 87% identity to LHCSR3. In contrast to PSBS, LHCSR proteins are chlorophyll a/b and xanthophyll-binding proteins.13 It has been demonstrated that LHCSR3 is a strong quencher of Chl excited states, exhibiting very fast fluorescence decay, with lifetimes below 100 ps. Hence they likely operate mainly in photoprotection.13 Gene and protein expression studies and mircoarray analyses revealed that expression of lhcsr genes were induced under high light stress, phosphorus-, iron- and sulfurdeficiency.14-17 The strong induction in expression under stress is consistent with a role of these proteins in acclimation toward photo-oxidizing conditions. Indeed, the qE capacity of Chlamydomonas increases proportionally with the lightdependent increase of LHCSR3 protein expression. There is currently no evidence that PSBS besides LHCSR3 participates in the establishment of qE in C. reinhardtii, although expression of PSBS in the alga has been reported under nitrogen deficiency.18 The finding that PSBS is not involved in qE is remarkable given that 2 psbs genes are encoded in the C. reinhardtii genome. Importantly, also in diatoms, a LHCSR orthologous protein, LHCX, has been demonstrated to be crucial for efficient qE.19 In consequence the function of PSBS in
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photosynthetic electron transfer, in particular with cyclic electron flow (CEF), has been an important constraint in the evolution of the PSBS dependent qE mechanism in land plants.21 Notably, PSBS is designated as PSII subunit in vascular plants. In Chlamydomonas, there is emerging evidence that LHCSR3 may interact with PSII as well as PSI.
LHCSR3 interacts with PSII and PSI
Figure 1. Model for the interaction between PSBR and LHCSR3 within PSII-LHCII supercomplexes in C. reinhardtii under high-light conditions. Schematic top view from lumenal side shows suggested locations of PSBR, LHCSR3 and the extrinsic proteins PSBO, PSBP and PSBQ. Highlight acclimated C. reinhardtii cells induce LHCSR3 expression. LHCSR3 binds to the PSII-LHCII supercomplex (C2S2M2L2), probably through the PSII subunit PSBR (2 possible binding scenarios are indicated – dashed lines), which might allow energetic coupling required for dissipating excess absorbed energy.
the alga remains unclear. Interestingly, while C. reinhardtii only uses LHCSR3 for driving qE, the moss Physcomitrella patens which codes for psbs and lhcsr genes utilizes both types of regulatory proteins to operate qE.20 This sheds an interesting light on the evolution of terrestrial plants pointing to the fact that land plants evolved a novel PSBSdependent qE mechanism before losing the ancestral LHCSRspecific mechanism found in algae. It is also of note that qE is constitutive in vascular plants, whereas, as mentioned above, it is induced upon acclimation to high-light in green algae.9 It has been suggested that the tight functional link between qE and
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It is commonly accepted that the PSII antenna is the site where quenching occurs.22 As LHCSR3 exhibits pigment-binding abilities and builds stable as well as specific complexes with chlorophyll and xanthophyll13 it appears to be directly involved in the quenching process at PSII-LHCII supercomplexes.11 However, the precise molecular arrangement of the quenching site and the mechanistic interaction of LHCSR3 with PSII still is largely unknown. A recent study in Chlamydomonas reported that LHCSR3 binds to the PSII-LHCII supercomplex, in which 3 LHCII trimers are attached to each side of the core (C2S2M2L2; 23), forming a PSII-LHCII-LHCSR3 supercomplex.11 This complex exhibited a low-fluorescence (energy-dissipative) state at pH 5.5 with LHCSR3 being essential for a high quenching capacity.11 In this current model LHCSR3 within the complex functions as pH signal transductor via a conformational change in a C-terminal subdomain of the protonated protein (upon lumenal acidification), thereby reversibly switching the supercomplex from a light-harvesting to a photo-protective state. Recently, our group obtained evidence that in high-light acclimated C. reinhardtii the PSII subunit PSBR is crucial for the binding of LHCSR3 to PSII-LHCII supercomplexes as well as for efficient NPQ (qE) and the integrity of the PSII-LHCIILHCSR3 supercomplex. Our study additionally suggests that PSBR contributes in forming a recognition site for LHCSR3 within the PSII-LHCII supercomplex, whereas the binding of LHCSR3 to the PSBR site might allow energetic interaction between LHCSR3, the PSII core and its associated LHCII complexes for qE quenching (Fig. 1). Furthermore, we proposed that LHCSR3 is rather linked to the PSII core complex than to the mobile LHCBM fraction due to the observation that PSBR and LHCSR3 co-migrate with PSII core subunits.24 Isolated pigment-protein complexes from high-light acclimated wild type C. reinhardtii showed co-migration of LHCSR3 with PSII as well as PSI24 in accordance with a previous report by Allorent and co-workers25 which suggested that LHCSR3 could migrate between PSII and PSI during state 1 to state 2 transition. Even though functional properties of LHCSR3 at PSI are unknown, such a targeted migration of LHCSR3 might be a tool for modulating the level of energy quenching in PSII complexes as well as in PSI complexes. New data indicated that LHCSR3 also interacts with PSI-LHCI-FNR supercomplexes 26 and that the absence of LHCSR3 accelerates photoinhibition of PSI under conditions where PSI in prone to degradation due to deletion of PGRL1.26 Now, what determines the interaction of LHCSR3 with other photosynthetic proteins and protein
Plant Signaling & Behavior
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Figure 2. (A) Peptide sequence alignment (ClustalW) of algal LHCSR-like proteins. Phosphorylation sites of LHCSR3 from C. reinhardtii (underlined) are indicated by arrows. (B) MS-based label free quantitation of peptides released from trypsin-digested thylakoid membranes (log10 scale). No quantitation data for peptides harbouring phosphorylated T250 and S258 is available.
complexes? A key in understanding could be the phosphorylation status of LHCSR3, which might modulate protein-protein-interaction properties. LHCSR3 is phosphorylated at the serine/threonine residues S26, S28, T32, T33 and T39 (Fig. 2A). The
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comparison of polypeptide sequences of LHCSR-like proteins from various algal species revealed that these phosphorylation sites are located in a region of low sequence conservation. Only few LHCSR proteins, namely those of Physcomitrella patens and
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Volvox, show a similar accumulation of potential acceptor sites. Hence, N-terminal phosphorylation of LHCSR-like proteins may not occur in all algae. However, it must be taken into account that the presence of signal peptide sequences may have interfered with the sequence alignment process. Therefore it cannot be ruled out that N-terminal phosphorylation does occur in other species, but at residues not aligning with those of LHCSR3 from Chlamydomonas. In line, all LHCSR protein sequences shown (Fig. 2A) possess S and T residues in their N-terminal parts. According to quantitative analyses 60% of the phosphorylatable residues S26, S28, T32, T33 and T39 are indeed phosphorylated under high light conditions.26 Notably these N-terminal residues are phosphorylated in an STT7-dependent manner.26 Moreover, a STT7-independent phosphorylation was found at a threonine residue (T185) in the C-terminal region that is not proteotypic and shared between LHCSR1 and LHCSR3, demonstrating that a second chloroplast kinase, other than STT7, is phosphorylating LHCSR protein(s) at their C-terminal part (Fig. 2). At the same position LHCSR-like proteins from other algae feature either potentially phosphorylatable residues like LHCSR3 or glutamic acid. Since glutamate possesses similar physical characteristics like phosphoserine and phosphothreonine, it seems reasonable to speculate that these residues play important roles in LHCSR3 function and/or stability. However, it remains to be elucidated if phosphorylation actually takes place in all LHCSR-like proteins and under which conditions the References 1. Whatley FR, Arnon DI, Tagawa K. Separation of light and dark reactions in electron transfer during photosynthesis. Proc Nat Acad Sci U S A 1963; 49:266-&; PMID:14000214; http://dx.doi.org/10.1073/pnas. 49.2.266 2. Koziol AG, Borza T, Ishida K, Keeling P, Lee RW, Durnford DG. Tracing the evolution of the light-harvesting antennae in chlorophyll a/b-containing organisms. Plant Physiol 2007; 143:1802-16; PMID:17307901; http://dx.doi.org/10.1104/pp.106. 092536 3. Wolfe GR, Cunningham FX, Durnford D, Green BR, Gantt E. Evidence for a common origin of chloroplasts with light-harvesting complexes of different pigmentation. Nature 1994; 367:566-8; http://dx.doi.org/ 10.1038/367566a0 4. Li XP, Bjorkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 2000; 403:391-5; PMID:10667783; http://dx.doi.org/10.1038/35000131 5. Li XP, Gilmore AM, Caffarri S, Bassi R, Golan T, Kramer D, Niyogi KK. Regulation of photosynthetic light harvesting involves intrathylakoid lumen pH sensing by the PsbS protein. J Biol Chem 2004; 279:22866-74; PMID:15033974; http://dx.doi.org/ 10.1074/jbc.M402461200 6. Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi R. Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant. J Biol Chem 2002; 277:22750-8; PMID:11934892; http://dx.doi.org/10.1074/jbc. M200604200 7. Demmig-Adams B, Adams WW 3rd. Harvesting sunlight safely. Nature 2000; 403:371, 3-4; http://dx.doi. org/10.1038/35000315 8. Demmig-Adams B, Gilmore AM, Adams WW 3rd. Carotenoids 3: in vivo function of carotenoids in
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9.
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ability to perform selective (transient) phosphorylation to ‘mimic’ glutamate is advantageous. For LHCSR1 no other nonproteotypic phosphorylated peptides were found, suggesting that LHCSR1, in contrast to LHCSR3, might be only phosphorylated at its C-terminus. Interestingly, in a STT7-deletion mutant, the amount of LHCSR3 that associated with PSI-LHCI-FNR complexes was significantly increased as compared to wild type.26 Thus suggesting, that the phosphorylation status of LHCSR3 is critical for the association with PSI supercomplexes. In wild type cells, deactivation of STT7 kinase activity due to an exceedingly reduced redox poise in the chloroplast stroma 27 could lead to an increase in the amount of non-phosphorylated LHCSR3 thereby promoting association with PSI supercomplexes which in turn results in enhanced photoprotection of PSI.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. HI 739/7.2 in FOR964 and HI 39/8.1 to M. H.) as well as the Chinese Scholarship Council (to H.X.).
higher plants. Faseb J 1996; 10:403-12; PMID:8647339 Peers G, Truong TB, Ostendorf E, Busch A, Elrad D, Grossman AR, Hippler M, Niyogi KK. An ancient lightharvesting protein is critical for the regulation of algal photosynthesis. Nature 2009; 462:518-21; PMID:19940928; http://dx.doi.org/10.1038/nature08587 Petroutsos D, Busch A, Janssen I, Trompelt K, Bergner SV, Weinl S, Holtkamp M, Karst U, Kudla J, Hippler M. The chloroplast calcium sensor CAS is required for photoacclimation in Chlamydomonas reinhardtii. Plant Cell 2011; 23:2950-63; PMID:21856795; http://dx.doi.org/10.1105/tpc.111.087973 Tokutsu R, Minagawa J. Energy-dissipative supercomplex of photosystem II associated with LHCSR3 in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 2013; 110:10016-21; PMID:23716695; http://dx.doi. org/10.1073/pnas.1222606110 Gagne G, Guertin M. The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis. Plant Mol Biol 1992; 18:429-45; PMID:1371402; http://dx. doi.org/10.1007/BF00040659 Bonente G, Ballottari M, Truong TB, Morosinotto T, Ahn TK, Fleming GR, Niyogi KK, Bassi R. Analysis of LhcSR3, a protein essential for feedback de-excitation in the green alga Chlamydomonas reinhardtii. PLoS Biol 2011; 9:e1000577; PMID:21267060; http://dx. doi.org/10.1371/journal.pbio.1000577 Zhang Z, Shrager J, Jain M, Chang CW, Vallon O, Grossman AR. Insights into the survival of Chlamydomonas reinhardtii during sulfur starvation based on microarray analysis of gene expression. Eukaryot Cell 2004; 3:1331-48; PMID:15470261; http://dx.doi.org/ 10.1128/EC.3.5.1331-1348.2004 Moseley JL, Chang CW, Grossman AR. Genomebased approaches to understanding phosphorus deprivation responses and PSR1 control in Chlamydomonas
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reinhardtii. Eukaryot Cell 2006; 5:26-44; PMID:16400166; http://dx.doi.org/10.1128/EC.5.1. 26-44.2006 Im CS, Zhang Z, Shrager J, Chang CW, Grossman AR. Analysis of light and CO(2) regulation in Chlamydomonas reinhardtii using genome-wide approaches. Photosynth Res 2003; 75:111-25; PMID:16245082; http://dx.doi.org/10.1023/A:1022800630430 Naumann B, Busch A, Allmer J, Ostendorf E, Zeller M, Kirchhoff H, Hippler M. Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii. Proteomics 2007; 7:3964-79; PMID:17922516; http://dx.doi.org/10.1002/pmic. 200700407 Miller R, Wu G, Deshpande RR, Vieler A, Gartner K, Li X, Moellering ER, Z€auner S, Cornish AJ, Liu B, et al. Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 2010; 154:1737-52; PMID:20935180; http://dx.doi.org/ 10.1104/pp.110.165159 Bailleul B, Rogato A, de Martino A, Coesel S, Cardol P, Bowler C, Falciatore A, Finazzi G. An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light. Proc Natl Acad Sci U S A 2010; 107:18214-9; PMID:20921421; http://dx.doi.org/10.1073/pnas. 1007703107 Alboresi A, Gerotto C, Giacometti GM, Bassi R, Morosinotto T. Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization. Proc Natl Acad Sci U S A 2010; 107:11128-33; PMID:20505121; http://dx.doi.org/10.1073/pnas. 1002873107 Kukuczka B, Magneschi L, Petroutsos D, Steinbeck J, Bald T, Powikrowska M, Fufezan C, Finazzi G, Hippler M. Proton gradient regulation5-like1-mediated
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cyclic electron flow is crucial for acclimation to anoxia and complementary to nonphotochemical quenching in stress adaptation. Plant Physiol 2014; 165:1604-17; PMID:24948831; http://dx.doi.org/10.1104/pp.114. 240648 22. Ruban AV, Berera R, Ilioaia C, van Stokkum IH, Kennis JT, Pascal AA, van Amerongen H, Robert B, Horton P, van Grondelle R. Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 2007; 450:575-8; PMID:18033302; http://dx. doi.org/10.1038/nature06262 23. Tokutsu R, Kato N, Bui KH, Ishikawa T, Minagawa J. Revisiting the supramolecular organization of photosystem II in Chlamydomonas reinhardtii. J Biol Chem
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2012; 287:31574-81; PMID:22801422; http://dx.doi. org/10.1074/jbc.M111.331991 24. Xue H, Tokutsu R, Bergner SV, Scholz M, Minagawa J, Hippler M. 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. Plant Physiol 2015; 167:1566-78; PMID:25699588; http://dx.doi.org/10.1104/pp.15.00094 25. Allorent G, Tokutsu R, Roach T, Peers G, Cardol P, Girard-Bascou J, Seigneurin-Berny D, Petroutsos D, Kuntz M, Breyton C, et al. A dual strategy to cope with high light in Chlamydomonas reinhardtii. Plant Cell 2013; 25:545-57; PMID:23424243; http://dx.doi. org/10.1105/tpc.112.108274
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26. Bergner SV, Scholz M, Trompelt K, Barth J, Gabelein P, Steinbeck J, Xue H, Clowez S, Fucile G, Goldschmidt-Clermont M, et al. State transition7-dependent phosphorylation is modulated by changing environmental conditions and its absence triggers remodeling of photosynthetic protein complexes. Plant Physiol 2015; 168:615-34; PMID:25858915; http:// dx.doi.org/10.1104/pp.15.00072 27. Lemeille S, Willig A, Depege-Fargeix N, Delessert C, Bassi R, Rochaix JD. Analysis of the chloroplast protein kinase Stt7 during state transitions. PLoS Biol 2009; 7: e45; PMID:19260761; http://dx.doi.org/10.1371/ journal.pbio.1000045
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Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii Huidan Xuey, Sonja Verena Bergnery, Martin Scholz, and Michael Hippler* Institute of Plant Biology and Biotechnology; University of M€ unster; M€ unster, Germany y
These authors equally contributed to this work.
Keywords: Chlamydomonas reinhardtii, excess light to heat dissipation, light-harvesting, LHCSR3, photosynthesis
Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photooxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii.
LHCSR3 is required for qE in algae and mosses Photosynthetic organisms convert solar energy into chemical energy by employing a series of reactions in and at the thylakoid membrane resulting in light-dependent water oxidation, NADP reduction and ATP formation.1 Hereby light is captured by the intrinsic chlorophyll a antenna systems of photosystem I (PSI) and photosystem II (PSII) as well as via associated chlorophyll a/b binding light harvesting complexes (LHC). The LHC superfamily is a monophyletic group and includes high light-induced proteins (HLIPs), stress-enhanced proteins (SEPs), PSBS and early light-inducible protein (ELIP).2,3 It is well accepted that LHC proteins not only functionally collect light energy for photosynthesis but also protect plants and algae from excess light. In such conditions, specialized light-harvesting proteins facilitate de-excitation of excess photons to avoid photodamage in high light. The fastest response to excess light is provided by a mechanism called non-photochemical quenching (NPQ). An important constituent of NPQ is the energy-dependent quenching (qE), which regulates the thermal dissipation of excess absorbed light energy. To become functional it requires the acidification of the thylakoid lumen via photosynthetic electron transfer. Thus the qE mechanism is functionally coupled to photosynthetic electron transfer and provides so efficient photo-protection. In *Correspondence to: Michael Hippler; Email: [email protected] Submitted: 05/26/2015; Accepted: 06/01/2015 http://dx.doi.org/10.1080/15592324.2015.1058462
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vascular plants, PSBS, a protein of the LHC protein superfamily having 4 transmembrane helices, is essential for efficient qE.4 PSBS functions as a lumenal pH sensor and regulates qE in vascular plants via protonation of 2 glutamate residues (Glu-122 and Glu-226), which both can be modified with dicyclohexylcarbodiimide (DCCD).5,6 Moreover, in vascular plants, acidification of the lumen activates the biochemical conversion of the light-harvesting violaxanthin to antheraxanthin and zeaxanthin via vioaxanthin de-epoxidase, that is required for effective qE.7,8 In green algae, the qE capacity is dependent on LHCSR3.9 Similar to vascular plants, also in green algae low lumenal pH is necessary for qE formation,10 which likely operates via protonation of acidic groups in LHCSR3, as LHCSR3 binds DCCD when associated to PSII-LHCII supercomplexes.11 LHCSR-like proteins (formerly named LI818-like or LHCX-like proteins) were originally identified as light-induced mRNA transcripts and are present in green algae, haptophytes, heterokonts and chromerida,12 but absent in vascular plants and red algae.2 There are 3 nuclear encoding lhcsr genes in Chlamydomonas reinhardtii, which are lhcsr1, lhcsr3.1 and lhcsr3.2.9 Both lhcsr3.1 and lhcsr3.2 encode an identical 259-amino-acid polypeptide. The other, paralogous gene lhcsr1 codes for a protein of 253 residues with 87% identity to LHCSR3. In contrast to PSBS, LHCSR proteins are chlorophyll a/b and xanthophyll-binding proteins.13 It has been demonstrated that LHCSR3 is a strong quencher of Chl excited states, exhibiting very fast fluorescence decay, with lifetimes below 100 ps. Hence they likely operate mainly in photoprotection.13 Gene and protein expression studies and mircoarray analyses revealed that expression of lhcsr genes were induced under high light stress, phosphorus-, iron- and sulfurdeficiency.14-17 The strong induction in expression under stress is consistent with a role of these proteins in acclimation toward photo-oxidizing conditions. Indeed, the qE capacity of Chlamydomonas increases proportionally with the lightdependent increase of LHCSR3 protein expression. There is currently no evidence that PSBS besides LHCSR3 participates in the establishment of qE in C. reinhardtii, although expression of PSBS in the alga has been reported under nitrogen deficiency.18 The finding that PSBS is not involved in qE is remarkable given that 2 psbs genes are encoded in the C. reinhardtii genome. Importantly, also in diatoms, a LHCSR orthologous protein, LHCX, has been demonstrated to be crucial for efficient qE.19 In consequence the function of PSBS in
Plant Signaling & Behavior
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photosynthetic electron transfer, in particular with cyclic electron flow (CEF), has been an important constraint in the evolution of the PSBS dependent qE mechanism in land plants.21 Notably, PSBS is designated as PSII subunit in vascular plants. In Chlamydomonas, there is emerging evidence that LHCSR3 may interact with PSII as well as PSI.
LHCSR3 interacts with PSII and PSI
Figure 1. Model for the interaction between PSBR and LHCSR3 within PSII-LHCII supercomplexes in C. reinhardtii under high-light conditions. Schematic top view from lumenal side shows suggested locations of PSBR, LHCSR3 and the extrinsic proteins PSBO, PSBP and PSBQ. Highlight acclimated C. reinhardtii cells induce LHCSR3 expression. LHCSR3 binds to the PSII-LHCII supercomplex (C2S2M2L2), probably through the PSII subunit PSBR (2 possible binding scenarios are indicated – dashed lines), which might allow energetic coupling required for dissipating excess absorbed energy.
the alga remains unclear. Interestingly, while C. reinhardtii only uses LHCSR3 for driving qE, the moss Physcomitrella patens which codes for psbs and lhcsr genes utilizes both types of regulatory proteins to operate qE.20 This sheds an interesting light on the evolution of terrestrial plants pointing to the fact that land plants evolved a novel PSBSdependent qE mechanism before losing the ancestral LHCSRspecific mechanism found in algae. It is also of note that qE is constitutive in vascular plants, whereas, as mentioned above, it is induced upon acclimation to high-light in green algae.9 It has been suggested that the tight functional link between qE and
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It is commonly accepted that the PSII antenna is the site where quenching occurs.22 As LHCSR3 exhibits pigment-binding abilities and builds stable as well as specific complexes with chlorophyll and xanthophyll13 it appears to be directly involved in the quenching process at PSII-LHCII supercomplexes.11 However, the precise molecular arrangement of the quenching site and the mechanistic interaction of LHCSR3 with PSII still is largely unknown. A recent study in Chlamydomonas reported that LHCSR3 binds to the PSII-LHCII supercomplex, in which 3 LHCII trimers are attached to each side of the core (C2S2M2L2; 23), forming a PSII-LHCII-LHCSR3 supercomplex.11 This complex exhibited a low-fluorescence (energy-dissipative) state at pH 5.5 with LHCSR3 being essential for a high quenching capacity.11 In this current model LHCSR3 within the complex functions as pH signal transductor via a conformational change in a C-terminal subdomain of the protonated protein (upon lumenal acidification), thereby reversibly switching the supercomplex from a light-harvesting to a photo-protective state. Recently, our group obtained evidence that in high-light acclimated C. reinhardtii the PSII subunit PSBR is crucial for the binding of LHCSR3 to PSII-LHCII supercomplexes as well as for efficient NPQ (qE) and the integrity of the PSII-LHCIILHCSR3 supercomplex. Our study additionally suggests that PSBR contributes in forming a recognition site for LHCSR3 within the PSII-LHCII supercomplex, whereas the binding of LHCSR3 to the PSBR site might allow energetic interaction between LHCSR3, the PSII core and its associated LHCII complexes for qE quenching (Fig. 1). Furthermore, we proposed that LHCSR3 is rather linked to the PSII core complex than to the mobile LHCBM fraction due to the observation that PSBR and LHCSR3 co-migrate with PSII core subunits.24 Isolated pigment-protein complexes from high-light acclimated wild type C. reinhardtii showed co-migration of LHCSR3 with PSII as well as PSI24 in accordance with a previous report by Allorent and co-workers25 which suggested that LHCSR3 could migrate between PSII and PSI during state 1 to state 2 transition. Even though functional properties of LHCSR3 at PSI are unknown, such a targeted migration of LHCSR3 might be a tool for modulating the level of energy quenching in PSII complexes as well as in PSI complexes. New data indicated that LHCSR3 also interacts with PSI-LHCI-FNR supercomplexes 26 and that the absence of LHCSR3 accelerates photoinhibition of PSI under conditions where PSI in prone to degradation due to deletion of PGRL1.26 Now, what determines the interaction of LHCSR3 with other photosynthetic proteins and protein
Plant Signaling & Behavior
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Figure 2. (A) Peptide sequence alignment (ClustalW) of algal LHCSR-like proteins. Phosphorylation sites of LHCSR3 from C. reinhardtii (underlined) are indicated by arrows. (B) MS-based label free quantitation of peptides released from trypsin-digested thylakoid membranes (log10 scale). No quantitation data for peptides harbouring phosphorylated T250 and S258 is available.
complexes? A key in understanding could be the phosphorylation status of LHCSR3, which might modulate protein-protein-interaction properties. LHCSR3 is phosphorylated at the serine/threonine residues S26, S28, T32, T33 and T39 (Fig. 2A). The
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comparison of polypeptide sequences of LHCSR-like proteins from various algal species revealed that these phosphorylation sites are located in a region of low sequence conservation. Only few LHCSR proteins, namely those of Physcomitrella patens and
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Volvox, show a similar accumulation of potential acceptor sites. Hence, N-terminal phosphorylation of LHCSR-like proteins may not occur in all algae. However, it must be taken into account that the presence of signal peptide sequences may have interfered with the sequence alignment process. Therefore it cannot be ruled out that N-terminal phosphorylation does occur in other species, but at residues not aligning with those of LHCSR3 from Chlamydomonas. In line, all LHCSR protein sequences shown (Fig. 2A) possess S and T residues in their N-terminal parts. According to quantitative analyses 60% of the phosphorylatable residues S26, S28, T32, T33 and T39 are indeed phosphorylated under high light conditions.26 Notably these N-terminal residues are phosphorylated in an STT7-dependent manner.26 Moreover, a STT7-independent phosphorylation was found at a threonine residue (T185) in the C-terminal region that is not proteotypic and shared between LHCSR1 and LHCSR3, demonstrating that a second chloroplast kinase, other than STT7, is phosphorylating LHCSR protein(s) at their C-terminal part (Fig. 2). At the same position LHCSR-like proteins from other algae feature either potentially phosphorylatable residues like LHCSR3 or glutamic acid. Since glutamate possesses similar physical characteristics like phosphoserine and phosphothreonine, it seems reasonable to speculate that these residues play important roles in LHCSR3 function and/or stability. However, it remains to be elucidated if phosphorylation actually takes place in all LHCSR-like proteins and under which conditions the References 1. Whatley FR, Arnon DI, Tagawa K. Separation of light and dark reactions in electron transfer during photosynthesis. Proc Nat Acad Sci U S A 1963; 49:266-&; PMID:14000214; http://dx.doi.org/10.1073/pnas. 49.2.266 2. Koziol AG, Borza T, Ishida K, Keeling P, Lee RW, Durnford DG. Tracing the evolution of the light-harvesting antennae in chlorophyll a/b-containing organisms. Plant Physiol 2007; 143:1802-16; PMID:17307901; http://dx.doi.org/10.1104/pp.106. 092536 3. Wolfe GR, Cunningham FX, Durnford D, Green BR, Gantt E. Evidence for a common origin of chloroplasts with light-harvesting complexes of different pigmentation. Nature 1994; 367:566-8; http://dx.doi.org/ 10.1038/367566a0 4. Li XP, Bjorkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 2000; 403:391-5; PMID:10667783; http://dx.doi.org/10.1038/35000131 5. Li XP, Gilmore AM, Caffarri S, Bassi R, Golan T, Kramer D, Niyogi KK. Regulation of photosynthetic light harvesting involves intrathylakoid lumen pH sensing by the PsbS protein. J Biol Chem 2004; 279:22866-74; PMID:15033974; http://dx.doi.org/ 10.1074/jbc.M402461200 6. Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi R. Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant. J Biol Chem 2002; 277:22750-8; PMID:11934892; http://dx.doi.org/10.1074/jbc. M200604200 7. Demmig-Adams B, Adams WW 3rd. Harvesting sunlight safely. Nature 2000; 403:371, 3-4; http://dx.doi. org/10.1038/35000315 8. Demmig-Adams B, Gilmore AM, Adams WW 3rd. Carotenoids 3: in vivo function of carotenoids in
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ability to perform selective (transient) phosphorylation to ‘mimic’ glutamate is advantageous. For LHCSR1 no other nonproteotypic phosphorylated peptides were found, suggesting that LHCSR1, in contrast to LHCSR3, might be only phosphorylated at its C-terminus. Interestingly, in a STT7-deletion mutant, the amount of LHCSR3 that associated with PSI-LHCI-FNR complexes was significantly increased as compared to wild type.26 Thus suggesting, that the phosphorylation status of LHCSR3 is critical for the association with PSI supercomplexes. In wild type cells, deactivation of STT7 kinase activity due to an exceedingly reduced redox poise in the chloroplast stroma 27 could lead to an increase in the amount of non-phosphorylated LHCSR3 thereby promoting association with PSI supercomplexes which in turn results in enhanced photoprotection of PSI.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. HI 739/7.2 in FOR964 and HI 39/8.1 to M. H.) as well as the Chinese Scholarship Council (to H.X.).
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