Molecular Mechanisms of Photoadaptation of Photosystem I ... · Dissecting the molecular mechanisms...

Molecular Mechanisms of Photoadaptation ofPhotosystem I Supercomplex from an EvolutionaryCyanobacterial/Algal Intermediate1[OPEN]

Patrycja Haniewicz,a Mateusz Abram,a,b Lukáš Nosek,c Joanna Kirkpatrick,d Eithar El-Mohsnawy,e,f

Julian D. Janna Olmos ,a,b Roman Kou�ril,c and Joanna M. Kargula,2

aSolar Fuels Laboratory, Center of New Technologies, University of Warsaw, 02-097 Warsaw, PolandbFaculty of Biology, University of Warsaw, 02-096 Warsaw, PolandcCentre of the Region Haná for Biotechnological and Agricultural Research, Department of Biophysics, Facultyof Science, Palacký University, 783 71 Olomouc, Czech RepublicdLeibniz Institute on Aging-Fritz Lipmann Institute, 07745 Jena, GermanyeBotany Department, Faculty of Science, Kafrelsheikh University, 33516, Kafr El-Sheikh, EgyptfPlant Biochemistry, Faculty of Biology and Biotechnology, Ruhr University, D-44780 Bochum, Germany

ORCID IDs: 0000-0003-0492-4625 (M.A.); 0000-0003-1622-129X (L.N.); 0000-0002-4905-5235 (J.D.J.O.); 0000-0003-1410-1905 (J.M.K.).

The monomeric photosystem I-light-harvesting antenna complex I (PSI-LHCI) supercomplex from the extremophilic redalga Cyanidioschyzon merolae represents an intermediate evolutionary link between the cyanobacterial PSI reaction centerand its green algal/higher plant counterpart. We show that the C. merolae PSI-LHCI supercomplex is characterized byrobustness in various extreme conditions. By a combination of biochemical, spectroscopic, mass spectrometry, and electronmicroscopy/single particle analyses, we dissected three molecular mechanisms underlying the inherent robustness of theC. merolae PSI-LHCI supercomplex: (1) the accumulation of photoprotective zeaxanthin in the LHCI antenna and the PSIreaction center; (2) structural remodeling of the LHCI antenna and adjustment of the effective absorption cross section; and(3) dynamic readjustment of the stoichiometry of the two PSI-LHCI isomers and changes in the oligomeric state of the PSI-LHCI supercomplex, accompanied by dissociation of the PsaK core subunit. We show that the largest low light-treated C.merolae PSI-LHCI supercomplex can bind up to eight Lhcr antenna subunits, which are organized as two rows on the PsaF/PsaJ side of the core complex. Under our experimental conditions, we found no evidence of functional coupling of thephycobilisomes with the PSI-LHCI supercomplex purified from various light conditions, suggesting that the putativeassociation of this antenna with the PSI supercomplex is absent or may be lost during the purification procedure.

Extremophiles have evolved the remarkable strat-egies that allow them to thrive beyond some dauntingphysical and chemical limits of life on Earth. It isimportant to understand the molecular mechanismsthat define these limits of life under extreme condi-tions. Dissecting the molecular mechanisms ofadaptation to these challenging environmental con-ditions is crucial for understanding how life mayhave evolved and survived in the early history of ourplanet.

The emergence of oxygenic photosynthesis in cya-nobacteria over 2.5 billion years ago is often dubbedthe Big Bang of evolution (Barber, 2004), as it gaverise to an aerobic atmosphere and protective ozonelayer and allowed efficient aerobic cellular respira-tion and the colonization of Earth’s surface by meta-zoan life. As such, it triggered fundamental biospherechanges on an unprecedented scale. In natural pho-tosynthesis, the absorption of two quanta of lighttriggers the primary charge separation in the reactioncenters (RCs) of PSII and PSI followed by the vectorialelectron flow from PSII to PSI via the cytochrome b6 fcomplex, with the concomitant release of protons and

molecular oxygen. With the input of four photons,initially absorbed by chlorophyll (Chl) molecules, thecatalytic metal center of PSII accumulates four oxi-dizing equivalents required to produce a dioxygenmolecule from two substrate water molecules (Babcocket al., 1989; Kargul and Barber, 2011). Concomitantly,PSI upon absorption of the second photon providesenergy-rich electrons to reduce the final acceptors fer-redoxin and NADP+ to NADPH via ferredoxin-NADPreductase.

The photosynthetic apparatus of extremophilicmicroalgae has gained considerable interest due tothe exceptionally high enzymatic stability and activ-ity of its photoelectroactive components, makingthem attractive for numerous applications in fieldsranging from structural biology (Klukas et al., 1999;Jordan et al., 2001; Zouni et al., 2001; Kamiya andShen, 2003; Ferreira et al., 2004; Loll et al., 2005;Adachi et al., 2009; Guskov et al., 2009; Amunts et al.,2010; Umena et al., 2011; Suga et al., 2015; Ago et al.,2016) to biotechnology (León-Bañares et al., 2004) andbiophotovoltaics (Krassen et al., 2009; Iwuchukwuet al., 2010; Utschig et al., 2011; Kargul et al., 2012;

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http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-1622-129X

http://orcid.org/0000-0003-1622-129X

http://orcid.org/0000-0003-1622-129X

http://orcid.org/0000-0003-1622-129X

http://orcid.org/0000-0003-1622-129X

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0003-1410-1905

http://orcid.org/0000-0003-1410-1905

http://orcid.org/0000-0003-1410-1905

http://orcid.org/0000-0003-1410-1905

http://orcid.org/0000-0003-1410-1905

http://orcid.org/0000-0003-0492-4625

http://orcid.org/0000-0003-1622-129X

http://orcid.org/0000-0002-4905-5235

http://orcid.org/0000-0003-1410-1905

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Mershin et al., 2012; Gordiichuk et al., 2014; Ocakogluet al., 2014; Janna Olmos and Kargul, 2015; Olmoset al., 2017; Szalkowski et al., 2017). These applica-tions stemmed from the use of robust photosystemspurified to homogeneity from thermophilic prokar-yotic cyanobacteria or eukaryotic thermoacidophilicred microalgae. On the other hand, extremophilic redmicroalgae have been the favorite model organismsin which to study the evolution of fundamental pro-cesses of cell division and intracellular transport(Kuroiwa, 1998; Kuroiwa et al., 1998; Miyagishimaet al., 2003; Nishida et al., 2003) as well as, very re-cently, the evolution and function of the photosyn-thetic apparatus through structural, genomic, biochemical,spectroscopic, and mass spectrometric approaches(Adachi et al., 2009; Vanselow et al., 2009; Busch et al.,2010; Krupnik et al., 2013; Nilsson et al., 2014; Agoet al., 2016).

The photosynthetic apparatus of the red thermoa-cidophilic microalga Cyanidioschyzon merolae hasgained considerable interest, due to the unique evo-lutionary positioning of this species near the root ofthe red algal lineage that forms a basal group withinthe eukaryotes and diverged ;1.3 billion years agowithin the most ancient algal order of Cyanidiales(Nozaki et al., 2003; Reeb and Bhattacharya, 2010). Itis considered as an evolutionary intermediate linkbetween the photosynthetic apparatus of prokaryoticcyanobacteria and that of the eukaryotic phototrophsof the green lineage (Ohta et al., 2003; Busch andHippler, 2011; Kargul et al., 2012). As such, it com-bines several prokaryotic and eukaryotic structuraltraits. In particular, PSII displays predominantly

prokaryotic and some eukaryotic features. The pro-karyotic characteristics include the presence ofcyanobacterial-like phycobilisomes (PBSs), function-ing as the peripheral light-harvesting antenna, aswell as the presence of cyanobacterial subunits sta-bilizing the catalytic center of PSII, PsbV, and PsbU inaddition to the evolutionarily conserved PsbO sub-unit. The fourth subunit unique to red algae that ispositioned within the oxygen-evolving complex isthe PsbQ9 protein (Krupnik et al., 2013; Ago et al.,2016).

The red algal PSI-LHCI supercomplex is reminis-cent of its higher plant and green algal counterpartsin that it comprises the monomeric RC core complexcomposed of 13 subunits (PsaA–PsaF and PsaI–PsaO;Jensen et al., 2007; Vanselow et al., 2009) and is as-sociated with an asymmetrically located, crescent-like peripheral light-harvesting antenna complex(LHCI) composed of a variable number of Chla-binding Lhcr subunits, depending on the species (Tanet al., 1997; Busch et al., 2010; Thangaraj et al., 2011;Tian et al., 2017a). The analysis of the Galdieria sul-phuraria plastid genome suggests that the red algalPSI may have evolved even earlier than the present-day cyanobacterial, green algal, and higher plantcounterparts (Vanselow et al., 2009). The interestingfeatures of the red algal PSI are the retention of thecyanobacterial PsaM subunit and the lack of higherplant and green algal PsaH and PsaG subunits im-plied in both the docking of the mobile LHCII an-tenna during state transitions and the formation ofthe LHCI belt, respectively (Kargul et al., 2012).Moreover, the chimeric nature of the two core subu-nits PsaF and PsaL, accommodating both cyano-bacterial and higher plant-like structural domains,further supports the evolutionarily intermediatecharacter of the red algal PSI-LHCI supercomplex(Busch and Hippler, 2011; Kargul et al., 2012).

Recently, we provided, to our knowledge, the firstdirect evidence that the C. merolae PSII complexemploys two distinct molecular mechanisms of pho-toprotection upon exposure to high light (HL). Theseare the accumulation of the carotenoid zeaxanthin(Zea) in thylakoids and dimeric PSII complexes to-gether with a reversible RC-based nonphotochemicalquenching that is triggered by the acidification ofthylakoid lumen upon exposure to HL intensities(Krupnik et al., 2013). Both features are likely toprovide the basis for the remarkable robustness andsustained high oxygen-evolving activity of theC. merolae PSII across a wide range of extreme light,temperature, and pH conditions (Krupnik et al.,2013). In this study, we extended the mechanistic andstructural investigation of the extremophilic red algalphotosynthetic apparatus to the second photosystemof C. merolae, the PSI-LHCI supercomplex, in order togain an insight into the molecular basis of the ex-ceptional robustness of this complex upon its expo-sure to various extreme conditions. We provideseveral lines of evidence that the high photochemical

1 J.M.K., P.H., and M.A. acknowledge support from the PolishNational Science Centre (OPUS grant no. UMO-2014/15/B/NZ1/00975 to J.M.K.). Part of this work was also supported by the PolishMinistry of Science and Higher Education and the European ScienceFoundation within the Eurocores/EuroSolarFuels/Solarfueltandemprogramme (grant no. 844/N-ESF EuroSolarFuels/10/2011/0 toJ.M.K.). The work of L.N. and R.K. was supported by grant LO1204(Sustainable Development of Research in the Centre of the RegionHaná) from the National Program of Sustainability I from the Minis-try of Education, Youth, and Sports, Czech Republic. CIISB researchinfrastructure project LM2015043 funded by MEYS CR is gratefullyacknowledged for the financial support of the measurements at theCF Cryo-Electron Microscopy and Tomography. E.E.-M. acknowl-edges support from the Ministry of Higher Education Egypt.

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Joanna M. Kargul ([email protected]).

P.H., M.A., L.N., J.K., J.M.K., E.E.-M., and R.K. generated andprocessed the data and prepared the figures; J.D.J.O. contributed topurification and biochemical characterization of PSI and studies on itsinteraction with PBSs; J.K., J.M.K., P.H., and R.K. designed the exper-iments and analyzed and interpreted the data; J.M.K., R.K., and P.H.cowrote the article; J.M.K. conceived and coordinated the study.

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activity and stability of the C. merolae PSI-LHCIsupercomplex are due to the combined protectiveeffects of Zea accumulation within this complex,changes of the oligomeric state of this complex,as well as dynamic structural remodeling ofthe LHCI antenna upon exposure to changing lightconditions.

RESULTS AND DISCUSSION

Biochemical and Proteomic Characterization of the C.merolae PSI-LHCI Supercomplex Isolated from VaryingLight Regimes

Themain aim of our studywas to dissect themolecularmechanisms of photoadaptation of the C. merolae PSI-LHCI supercomplex. To this end, we purified the highlyhomogenous PSI-LHCI preparations by detergent-basedsolubilization of thylakoids obtained from cells grown infour distinct light regimes: low (LL; 35 mE m22 s21),medium (ML; 90 mEm22 s21), high (HL; 150 mE m22 s21),and extreme high (EHL; 350 mE m22 s21) white light ir-radiation. We applied three-step anion-exchange chro-matography (AEC) and size-exclusion chromatography(SEC) to obtain pure supercomplex preparations.Figure 1 shows the typical AEC chromatograms (identicalfor all four light regimes) and SDS-PAGE proteinprofiles, confirming that the all four PSI-LHCI sampleswere purified to homogeneity and contained thetypical core (e.g. PsaA/PsaB heterodimer and smallercore subunits) and Lhcr antenna subunits resolved astwo protein bands by SDS-PAGE and identified bymass spectrometry in all four PSI-LHCI preparations(Table I).The photochemical activity of all four PSI-LHCI

preparations (Fig. 1D) was in the range of 538 to 1,331mmol oxygen consumed mg21 Chl h21, with the highestvalue obtained for the EHL PSI-LHCI supercomplex.Overall, the activity of the red algal PSI-LHCI complexwas 1.3- to 3.2-fold higher than the activity of the tri-meric PSI complex purified from the thermophilic cy-anobacterium Thermosynechococcus elongatus, using ananalogous purification procedure and the same exper-imental conditions for the oxygen consumption mea-surement (Fig. 1D).To further probe the robustness of the C. merolae

PSI-LHCI supercomplex, we subjected the sampleobtained from ML to a wide range of extreme tem-perature, light, and pH conditions. The choice of theML sample was to avoid any putative detrimentaleffects of stress conditions prior to investigation ofthe robustness of the C. merolae PSI-LHCI super-complex. Figure 2 shows that the C. merolae PSI-LHCIsupercomplex retains most of its photochemical ac-tivity when exposed to light intensities as high as25,000 mE m22 s21, temperature up to 80°C, and a pHrange from 4 to 12. To our knowledge, this is the mostrobust PSI-LHCI supercomplex reported to date,even when compared directly with other relatively

stable PSI complexes such as the PSI trimer from thethermophilic cyanobacterium T. elongatus (Fig. 1D), fromwhich a near atomic x-ray structure was obtained(Jordan et al., 2001). The wide temperature tolerance ofisolated C. merolae PSII (Krupnik et al., 2013) and PSI-LHCI complexes (this study) is most likely the reason forthe remarkable wide temperature range tolerance of thisextremophilic alga, as shown in a recent study (Nikolovaet al., 2017).

The C. merolae PSI-LHCI supercomplex exists as amonomer, similar to its other eukaryotic counterparts(Busch et al., 2010; Drop et al., 2011; Mazor et al., 2015;Qin et al., 2015; Tian et al., 2017a). An interesting ob-servation is the presence of two native bands from thiscomplex that were identified by blue native (BN)-PAGE in LL/ML (50 mE m22 s21) C. merolae com-plexes, varying in the core and possibly LHCI subunitcomposition (Tian et al., 2017a). This prompted us toexamine the putative changes in the relative abun-dance of both isomers in response to changing illu-mination conditions. Figure 3 shows that the overallaverage size of the PSI-LHCI supercomplex decreasesgradually upon increasing light intensity (Fig. 3A). Inagreement with this observation, the relative abun-dance of the smaller and larger PSI-LHCI isomer(identified as band 1 and band 2, respectively, byBN-PAGE; Fig. 3B) changes dynamically in responseto varying light conditions, with the amount of thelarger form of this complex diminished severely in HLand EHL (Fig. 3, B and C) compared with LL and MLconditions. We observed an additional band (band 3;Fig. 3B) composed of the oligomeric forms of the PSI-LHCI supercomplex of varying abundance dependingon the light regime used, with the larger oligomersobserved preferentially under LL conditions.

To get an insight into the intriguing dynamicchanges of abundance of the three native bands, weexamined their precise subunit composition by massspectrometry. Most of the core subunits (except forPsaI) and all the three Lhcr subunits were identified inall the C. merolae PSI-LHCI isomers and oligomers atall the light intensities applied (Table I). The EHL band3 contained the same subunits as the EHL band 2,except for the PBS rod-core linker polypeptide, whichwas absent in the EHL oligomers (Table I). In contrastto the recent study of Tian et al. (2017a), the coresubunits PsaF and PsaO were associated with bothbands rather stably in all the light regimes. The PsaOsubunit is absent in the recent x-ray structures of thehigher plant PSI-LHCI supercomplex, and it has beenpostulated to be associated loosely with the PSI corecomplex (Mazor et al., 2015, 2017; Qin et al., 2015). Thepresence of PsaO in the C. merolae supercomplex,therefore, may reflect differences in its interaction withother core subunits in comparison with the higherplant complex. The PsaF subunit, apart from its well-established role as the docking site for the mobileelectron carriers (cytochrome c553 in C. merolae; for re-view, see Kargul et al., 2012), has been postulated asthe putative binding site for one of the LHCI subunits

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Photoadaptation of Robust C. merolae PSI



in the C. merolae PSI-LHCI supercomplex (Tian et al.,2017a), although this remains to be directly confirmedby structural data.

Of all the core subunits, the PsaK protein seems toexhibit the largest variability in its abundance in both

PSI-LHCI isomers and oligomers (Table I). In fact, thissubunit was missing in both LL bands as well as in alarger supercomplex isomer and oligomers isolatedfrom EHL conditions (Table I), suggesting that it is as-sociated rather loosely with the PSI core inC. merolae. In

Figure 1. Purification and biochemical characterization of LL,ML, HL, and EHL supercomplexes. A, AEC chromatogram from thefirst step of the C. merolae PSI-LHCI supercomplex purification procedure on a DEAE TOYOPEARL 650M column. Insets I and IIshow RTabsorption spectra of PSI and PSII fractions, respectively. B, AEC chromatogram from the second step of the C. merolaePSI-LHCI supercomplex purification procedure on aDEAE TOYOPEARL 650S column. Inset III shows a RTabsorption spectrum ofthe PSI-LHCI supercomplex after the secondAEC step. C, SDS-PAGE protein profiles of T. elongatus PSI trimer andC.merolae PSI-LHCI supercomplexes purified from four different light regimes (LL, ML, HL, and EHL). D, Photochemical activity of the C.merolae PSI-LHCI supercomplex purified from four different light regimes comparedwith the activity of the T. elongatus PSI trimerfrom ML.


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higher plants, the PsaK subunit interacts with PsaB onthe opposite side from a homologous PsaG subunit(absent in C. merolae) that forms the docking site for theLhca1 antenna protein (Mazor et al., 2015, 2017; Qinet al., 2015). The PsaK subunit has been postulatedto exist in two copies in another red microalga,G. sulphuraria (Vanselow et al., 2009). However, theprecise positioning of the second copy remains to beestablished. Therefore, the absence of PsaK in the largerisoform of the C. merolae PSI-LHCI supercomplex in LLand EHL conditions may reflect its close interactionwith one of the Lhcr subunits and their co-dissociationfollowing significant structural changes in PsaK. Suchconformational changes were postulated in the latestx-ray structure of the higher plant PSI-LHCI super-complex during light-induced remodeling of the LHCIantenna (Mazor et al., 2017).

Spectroscopic Investigation of the C. merolae Light-Harvesting Antenna Interaction with the PSI RC underVariable Light Conditions

Spectroscopic analysis of the LL, ML, HL, and EHLPSI-LHCI supercomplex samples showed the typicalabsorbance red maxima at 678.5 nm and 77K red-shifted emission peaks at 726 to 729.5 nm followingexcitation of Chla at 435 nm (Fig. 4, A and B, respec-tively), due to the presence of red Chls postulated toserve as intermediate energy traps for excitationstransferred to the P700 RC (Werst et al., 1992). Impor-tantly, the EHL PSI-LHCI sample showed a 3.5-nm redshift of the 77K emission maximum compared with LLsamples (Fig. 4B), most likely due to the accumulationof additional red Chls in the supercomplex upon ex-posure to extreme HL.

Table I. Mass spectrometry analysis of the BN-PAGE protein bands obtained from LL, ML, HL, and EHL C. merolae PSI-LHCI samples

Shown are the subunits identified in BN-PAGE bands in Fig. 3. The relative contributions are represented by crosses, where four crosses representproteins with iBAQ values (the sums of intensities of all tryptic peptides for each protein divided by the number of theoretically observable peptides) $1.75e10, three crosses $ 2.5e9, two crosses $ 5e8, and one cross $ 2.5e6. The negligible contribution of a protein is indicated with a minus sign,where the iBAQ value was , 2.5e6.





The presence of the red-absorbing Chls is a charac-teristic feature of PSI (Morosinotto et al., 2005; Wientjeset al., 2012; Croce and van Amerongen, 2013). The redenergy traps have been shown to slow down the rate ofenergy trapping in P700 through alteration of the ki-netics of excitation energy transfer pathways from theperipheral and core antennae to the RC of the eukary-otic and cyanobacterial PSI complexes (Croce et al.,2000; Gobets and van Grondelle, 2001; Ihalainen et al.,2002; Jennings et al., 2003; Gibasiewicz et al., 2005;Melkozernov et al., 2005; Engelmann et al., 2006;Snellenburg et al., 2013). The slower fluorescence decaykinetics evoked by the red traps is due mainly to theuphill energy transfer from the low-energy forms to thebulk and/or inner core Chl molecules (Croce et al.,2000; Jennings et al., 2003). The physiological role of redtraps is a matter of debate, although their plausible rolemay be to increase the PSI absorption cross section in ashaded environment or in the conditions favoring cyclicelectron flow around PSI (Rivadossi et al., 1999).

Red Chls have been localized mainly in the LHCIcomplex by thermal broadening spectroscopic analyses(Croce et al., 1996, 1998), which has been confirmeddirectly by the latest x-ray crystallography studies ofthe higher plant PSI-LHCI supercomplex (Mazor et al.,2015; Qin et al., 2015). However, it is not inconceivablethat they also may accumulate in the C. merolae PSI corecomplex in response to light stress, where they mayaffect the kinetics and pathways of energy transfer tothe P700 trap. Experiments to verify this hypothesis arecurrently under way.

A common observation was that, in all but the lastchromatographic step, a significant amount of PBSs,which serve as the peripheral antenna of PSII in C. mer-olae (Krupnik et al., 2013), was detected. To probe theputative functional coupling of the residual PBSs, iden-tified by mass spectrometry in the final pure PSI-LHCIsamples (Table I) with the PSI RC of C. merolae, wemeasured the emission spectra of all four final PSI-LHCIsamples upon their excitation at 600 nm, a wavelengththat selectively excites phycocyanin (a pigment presentin the PBS antenna). Figure 4C shows a small 722-nmemission peak corresponding to the PSI-LHCI super-complex. However, this peak was extremely small, dueto the lack of energy transfer between PBSs and the PSIRC at all the light regimes applied in this study.

To further probe the energy transfer between theperipheral antennae and the PSI RCs, we measured theexcitation spectra of all four ultra-pure PSI-LHCI sam-ples following excitation at 400 to 700 nm and recordingemission at 728 nm. The excitation spectra (Fig. 4D)corresponded to the pure PSI-LHCI supercomplexescontaining excitonically coupled LHCI antennae, albeit

Figure 2. Robustness of C. merolae PSI-LHCI in extreme conditions. A,Photochemical activity of the PSI-LHCI ML supercomplex in differentlight regimes. B, Photochemical activity of the PSI-LHCI supercomplex

exposed to various temperatures. C, Photochemical activity of the PSI-LHCI supercomplex in the pH range 4 to 12.


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with no energy transfer detected between PBSs and thePSI RC for all the PSI-LHCI samples analyzed.

Overall, we found no spectroscopic evidence of afunctional association of PBSs with the C. merolae PSIRC in all four light regimes applied in this study. Thisobservation is in contrast with the spectroscopic andproteomic results reported by Hippler and colleagues(Busch et al., 2010), who postulated the physical andfunctional association of a small fraction of PBSs with asubpool of the LL C. merolae PSI-LHCI supercomplex.This discrepancy is most likely due to the higher ho-mogeneity of the supercomplex samples obtained inthis study, because of the more stringent purificationprocedure, and the possible dissociation of the putativePBS fraction from the PSI-LHCI samples. However, wecannot exclude that such a functional association mayoccur in vivo and is lost during the purification proce-dure. In fact, during the purification of all four PSI-LHCIsamples, we observed a high degree of heterogeneity ofthe photosystem complexes, with some PSI-LHCI frac-tions enriched significantly in PBSs (data not shown).Whether they may form functionally coupled PBS-PSI-LHCI assemblies remains to be established.

Photoprotective Role of Zea Accumulating in the C.merolae PSI-LHCI Supercomplex upon Exposure to HL

In this study, we examined the putative photo-protective roles of carotenoids accumulating in theC. merolae cells and PSI-LHCI supercomplex prepara-tions upon exposure to various light conditions. Animportant issue was to determine the precise loci of thecarotenoid accumulation within the PSI-LHCI super-complex upon its exposure to various light regimes. Tothis end, we separated the LHCI antenna from the PSIcore particles by the detergent treatment followed by Sucgradient fractionation of the solubilized complexes(Fig. 5, A and B).

As expected, the isolated LHCI antenna displayedsignificant absorption in the 400-500 nm region and the77K Chla emission peak at 682-684 nm (Fig. 5, C and D),arising from the enrichment of this fraction with ca-rotenoids and the depletion of some red Chls (due to thedetergent treatment), respectively. The LHCI-depletedPSI fraction was characterized by a 4.5-6-nm blue shiftof the 77K Chla emission peak compared with the intactPSI-LHCI supercomplex (Fig. 5, C and D). This is mostlikely due to the loss of a fraction of the red Chls, whichare associated predominantly with the LHCI antenna(Mazor et al., 2015, 2017; Qin et al., 2015). Two mainpeaks of 722 and 675 nm were present in the 77Kemission spectra of the LHCI-depleted PSI fractions (for

Figure 3. Oligomeric state of the LL, ML, HL, and EHL C. merolaePSI-LHCI supercomplexes. A, SEC analysis of PSI-LHCI samplespurified from four different light regimes. Yellow, ML PSI trimer fromT. elongatus; blue, C. merolae LL PSI-LHCI; green, ML C. merolae

PSI-LHCI; gray, C. merolae HL PSI-LHCI; red, C. merolae EHL PSI-LHCI. B, BN-PAGE analysis of the C. merolae PSI-LHCI super-complex purified from four different light regimes. C, Densitometricanalysis of the bands from B.





ML samples; Fig. 5C). We interpret this observation bythe presence of tightly bound LHCI antenna whoseenergetic coupling with the PSI RC is destabilized dueto the detergent and freeze/thaw treatment duringseparation of the LHCI antenna. These spectroscopicresults indicate the heterogeneity of Lhcrs present in theC. merolae PSI-LHCI supercomplex in terms of varyingamounts of associated red Chls. The red peak of 722 nmin the LHCI-depleted core fraction points to the pres-ence of red Chls also in the core complex, similar to thecyanobacterial counterpart (Jordan et al., 2001). TheLhcr subunits were detected as single bands of ;20 kDby BN-PAGE (data not shown), suggesting that theyexist as monomers in contrast to the higher plant Lhcadimers (Mazor et al., 2017).

We then examined the putative changes in the ac-cumulation of carotenoids in whole cells exposed to allfour light regimes as well as purified intact PSI-LHCIsupercomplexes, LHCI-depleted PSI core particles,and the outer peripheral LHCI antenna complexes.To this end, we performed quantitative HPLC analy-sis of the pigments present in all of the above sam-ples to determine their molar ratios with respect to thetotal number of Chla molecules estimated in eachfraction (Tables II and III). Three main carotenoidswere identified in all the samples obtained fromfour different light regimes (Tables II and III;Supplemental Figs. S1 and S2): Zea, b-carotene (b-car),and b-cryptoxanthin (b-crypto [an intermediate of

Zea biosynthesis from b-car in C. merolae];Cunningham et al., 2007). Of all the carotenoidsidentified in our study, Zea accumulated in consid-erable amounts in both intact cells as well as theisolated PSI-LHCI, PSI core, and LHCI antennacomplexes in response to an increasing light intensity(Tables II and III; Supplemental Figs. S1 and S2).

Our results confirm the observations of Gantt andcolleagues (Cunningham et al., 1989), who showedthat, in the mesophilic unicellular red alga Porphyri-dium cruentum, the cellular content of Zea increasedwith growth irradiance, confirming a pivotal role forthis carotenoid in long-term light adaptation. Nev-ertheless, the molar ratios of Zea/Chla seem to be upto 3.3-fold higher in C. merolae cells (Table II) com-pared with the cells of P. cruentum, indicating thatthis carotenoid plays an important role in acclimationof the extremophilic red microalgae exposed to lowpH and high temperatures under variable light. Un-der such challenging conditions, the additional en-ergetic pressure may be exerted on the C. merolaephotosynthetic apparatus to cope with an increaseddemand on ATP to actively extrude protons from thecytoplasm. Therefore, the accumulation of photo-protective Zea may be one of the crucial strategies tomaintain the functional and structural integrity of thephotosynthetic apparatus and thylakoid membranesin this thermoacidophilic microalga. Interestingly,the b-car/Chl ratio showed only a minor increase, up

Figure 4. Spectroscopic characteriza-tion of the LL, ML, HL, and EHL C.merolae PSI-LHCI supercomplexes. A,RT absorption spectra of PSI-LHCIsamples purified from four differentlight regimes. Yellow, ML PSI trimerfrom T. elongatus; blue, C. merolae LLPSI-LHCI; green, ML C. merolae PSI-LHCI; gray, C. merolae HL PSI-LHCI;red, C. merolae EHL PSI-LHCI. B, 77Kemission spectra of PSI-LHCI com-plexes from four different light regimes,excited at 435 nm. C, 77K emissionspectra of complexes from four differ-ent light regimes, excited at 600 nm. D,77K excitation spectra of all the PSI-LHCI samples with emission recordedat 728 nm.


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to 20% for the EHL cells compared with LL cells, withthe exception of HL cell samples, for which a ratherlow b-car/Chl ratio was observed (Table II). Thesignificance of the latter observation is unknown atpresent, although it can be stipulated that the cellsexposed to HL conditions decrease the overall con-tent of b-car, perhaps due to the enhanced Zea syn-thesis from b-car that is triggered by light stress.

The relative amounts of total carotenoids estimatedin the intact PSI-LHCI supercomplex were 52, 39, 51,and 54 molecules (normalized to 14 Chls per Lhc sub-unit, as well as 8, 6, 5, and 4 Lhcrs per LL, ML, HL, andEHL PSI RC, and 98 Chls per PSI RC) for the LL, ML,HL, and EHL samples, respectively, indicating that theoverall amount of total carotenoids remained similar inthe PSI-LHCI supercomplexes pre-adapted to various

Figure 5. Biochemical and spectroscopic analyses of LHCI-depleted PSI core complexes and LHCI antennae from the LL,ML,HL,and EHL C. merolae PSI-LHCI supercomplexes. A, Separation of LHCI-depleted PSI core complexes (F2) and LHCI antennae (F1)by Suc gradient ultracentrifugation. B, Coomassie Blue-stained SDS-PAGE protein profiles for the fractions from the Suc gradientfrom LL, ML, HL, and EHL samples. C, Representative 77K emission spectra of PSI-LHCI, LHCI, and LHCI-depleted PSI excited at435 nm and emission at 728 nm following 400- to 700-nm excitation. For clarity, only ML emission spectra are shown. D, Tablewith the Qy band absorption andmain peaks in 77K emission spectra of PSI-LHCI, LHCI, and LHCI-depleted PSI fractions excitedat 435 nm and emission at 728 nm for all four light regimes.





light regimes (Table III). However, the relative amountof total carotenoids in the LHCI antenna complexesshowed a steady increase during adaptation of the cellsto excessive light. Thus, we identified 4, 6, 8, and12 carotenoids per Lhcr subunit from LL, ML, HL, andEHL LHCI fractions, respectively (Table III). The totalnumbers of carotenoids in theC.merolaeHLandEHLLHCIantenna complexes are significantly higher than for thehigher plant counterpart, where an average of 3-4 carote-noids were identified per Lhca subunit by x-ray crystal-lography (Qin et al., 2015; Mazor et al., 2017). Similarly, anaverage of 8 carotenoids per Lhcr was reported in the LL/ML LHCI fraction from the same alga (Tian et al., 2017a).

The quantification of the Zea/Chla molar ratiosallowed us to estimate the relative numbers of Zeamolecules as 28, 33, 36, and 45 per LL,ML,HL, and EHLPSI-LHCI supercomplex, respectively, following thesame normalization as above. The analogous estima-tion gives a total number of b-car molecules of 19, 4, 13,and 6 per LL, ML, HL, and EHL PSI-LHCI super-complex, respectively (Table III), indicating a 73% de-crease in the content of this carotenoid in the EHLC. merolae PSI-LHCI sample compared with the x-raystructure of the pea (Pisum sativum) PSI-LHCI super-complex, for which 22 b-car molecules were identified(Qin et al., 2015; Mazor et al., 2017). All the above datapoint to the conclusion that there may be significantstructural differences between the red algal PSI-LHCIsupercomplex and the higher plant counterpart withrespect to the intrinsic pigment organization during ad-aptation to excessive light, whereby Zea accumulation isaccompanied by a steady decrease in the b-car content.Significant differences in the pigment organisation andinteractions were also observed for the C. merolae PSI-LHCI complex and its cyanobacterial counterpart, asevidenced by the circular dichroism (CD) spectroscopy(Supplemental Fig. S3).

The comparison of pigment quantification data be-tween various samples prompted us to the conclusionthat the main locus for the accumulation of Zea in re-sponse to increasing light intensities is the LHCI an-tenna (Table III), in agreement with a previous study(Tian et al., 2017a). In the higher plant PSI-LHCIsupercomplex, 2% of the total amount of Zea wasdetected in the core complex, in addition to the majorityof this pigment being present in the LHCI antenna(Ballottari et al., 2014). Indeed, our LHCI-depleted PSI

complexes obtained from various light regimes con-tained 1-5 Zeamolecules (Table III), suggesting that thispigment also may evoke a light-harvesting role in theRC of PSI-LHCI supercomplex under light-limitingconditions. However, at this stage, it is impossible todetermine unequivocally whether these Zea moleculeswere associated exclusively with the pure PSI corecomplex, as this preparation still contained the residualamount of tightly bound, if excitonically uncoupled,Lhcr antenna subunits (Fig. 5C).

Our pigment quantification differs from the valuesobtained in the study by Tian et al. (2017a), probably dueto different light illumination and cell culture growthconditions used for obtaining the PSI-LHCI samples. Inthe previous study, 18molecules of Zeawere reported forthe LL/ML PSI-LHCI supercomplex of C. merolae, incontrast to 28-45 Zea molecules identified in this work.One important difference between our study and theabove-mentioned resultswas that fact thatwe applied thespecific light treatment to mid-log phase cultures to en-sure the complete penetration of light, in contrast to theprevious study, in which stationary cultures were usedfor all the biochemical and spectroscopic analyses.

The direct photoprotective role of Zea was proposed forthe higher plant PSI-LHCI supercomplex, where this ca-rotenoid, bound within the LHCI domain, was implicatedin a novel type of Zea-dependent nonphotochemicalquenching, involving the formation of the carotenoid rad-ical cations, that was coupled to an improved photo-stability of the PSI-LHCI supercomplex (Ballottari et al.,2014). It is important to emphasize that this phenomenonoccurred in the npq2 mutant of Arabidopsis thaliana thatconstitutively accumulatedZea and lacked violaxanthin,whereasHL-adaptedwild-type plants showed no Zea inthe PSI core complex (Ballottari et al., 2014). The postu-late of a direct role of Zea in photoprotection of thehigher plant PSI-LHCI supercomplex was recentlychallenged due to the similar time-resolved fluorescencekinetics of the PSI-LHCI complex in dark-adapted andHL conditions (Tian et al., 2017b), suggesting that thiscarotenoid may play a different role in photoprotectionof the photosynthetic apparatus than initially proposed(Ballottari et al., 2014).

A feasible physiological photoprotective role forZea may be to act as a direct antioxidant in scav-enging free radicals and singlet oxygen moleculesproduced in HL in the lipid phase of the thylakoid

Table II. Quantification of molar ratios of pigments in LL, ML, HL, and EHL C. merolae cells

Preparation Pigment and Light Regime

LL (35 mE) ML (90 mE) HL (150 mE) EHL (350 mE)

Zea/Chla 0.856 6 0.001 1.061 6 0.002 1.169 6 0.049 2.878 6 0.040b-Car/Chla 0.323 6 0.002 0.362 6 0.001 0.080 6 0.002 0.397 6 0.002b-Crypto/Chla 0.061 6 0.002 0.071 6 0.001 0.056 6 0.001 0.151 6 0.003

Molar ratios of pigments were calculated by integration of an area underneath the relevant peak and usingextinction coefficients, as described in “Materials and Methods.” SD values were calculated from one injec-tion from two independent preparations (n = 2).


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membranes, in the vicinity of PSII and PSI-LHCI mac-rodomains (Havaux et al., 2007; Johnson et al., 2007).Indeed, we observed a large amount of Zea also associ-ated with pure ML dimeric PSII samples from C. merolae(Krupnik et al., 2013), in addition to the presence of thiscarotenoid in purified PSI-LHCI supercomplex identifiedin this study. Free Zea molecules bound within theC. merolae PSI-LHCI and PSII complex-lipid interfacealso may protect the intrinsic lipid molecules, identified inthe x-ray structures of both red algal PSII and higherplant PSI (Mazor et al., 2015, 2017; Qin et al., 2015; Agoet al., 2016), from singlet oxygen-mediated peroxidation(Havaux et al., 2007). An additional structural role for

Zea may be to act as the rigidifying molecule (Gruszeckiand Strzałka, 2005), which could lead to better stabilityof C. merolae photosynthetic complexes at high tempera-tures and in HL conditions.

Antenna Remodeling of the C. merolae PSI-LHCISupercomplex under Varying Light Conditions

In Rhodophyta, the peripheral LHCI antenna systemis formed by Chla-binding Lhcr proteins whose numberper supercomplex is species dependent (Wolfe et al.,1994a, 1994b). Biochemical analyses showed the

Table III. Quantification of molar ratios of pigments in LL, ML, HL, and EHL C. merolae PSI-LHCI supercomplexes, LHCI antennae, and LHCI-depleted PSI core particles

Molar ratios of pigments were calculated by integration of an area underneath the relevant peak and using extinction coefficients, as described in“Materials and Methods.” Values in parentheses show the number of pigment molecules normalized to 210, 182, 168, and 154 Chls for the LL, ML, HL,and EHL PSI-LHCI supercomplexes, respectively. The calculations were performed assuming 14 Chls per Lhcr; eight, six, five, and four Lhcrs per LL, ML,HL, and EHL supercomplexes, respectively (see Table IV); 98 Chls per PSI core complex; as well as 112, 84, 70, and 56 Chls (Mazor et al., 2017) pereach respective LHCI complex. SD values were calculated from one to two injections from two independent preparations (n = 2).





presence of six Lhcr proteins in the red alga P. cruentum(Tan et al., 1997) and five Lhcr polypeptides in G. sul-phuraria (Marquardt et al., 2001). The nuclear genome ofC. merolae encodes three Lhcr polypeptides (Matsuzakiet al., 2004), which are present at various stoichiome-tries in the PSI-LHCI complex to form the asymmetricallylocated LHCI antenna belt, as confirmed by proteomicand electron microscopy coupled to single-particle anal-yses (Busch et al., 2010).

In this work, we tested the putative remodeling of theC. merolae LHCI antenna upon exposure to various lightintensities. Such remodeling of the LHCI antenna wasobserved for LL-grown cells of two extremophilic redmicroalgae, G. sulphuraria and Cyanidium caldarium;however, the precise number of the Lhcr subunits couldnot be determined (Gardian et al., 2007; Thangaraj et al.,2011). To gain an insight into the putative rearrangementof the peripheral light-harvesting antenna subunits as theadaptation mechanism to varying light conditions, wevisualized the negatively stained PSI-LHCI particles(purified from cells grown in LL, ML, and EHL condi-tions) by electron microscopy (Fig. 6) followed by single-particle analysis of their 2D projections. Single-particleaveraging of a large set of particles showed that theywere mostly present in a top-view position from thestromal side (Fig. 7). Single-particle analysis revealed twoto four specific forms of PSI-LHCI supercomplexeswhosepresence and relative abundance depend on the growthlight conditions. Under LL conditions, only two large andmedium forms are present (Fig. 7, A and B), with di-mensions of 19 3 17 nm and 19 3 15 nm, respectively.Under EHL conditions, the largest form of the PSI-LHCIsupercomplex is completely absent and two smallerforms can be identified, in addition to the medium sizeparticles obtained from LL and ML conditions (Fig. 7, Hand I), with dimensions of 19 3 14 nm and 17 3 15 nm,respectively. In ML conditions, all forms of the PSI-LHCIsupercomplex were present, indicating the largest struc-tural heterogeneity of this complex preparation.

To determine the location and estimate the number ofthe antenna subunits, we overlaid the x-ray structure of

the higher plant PSI-LHCI supercomplex (Mazor et al.,2017) onto the C. merolae PSI-LHCI supercomplex pro-jections. Figure 8 shows four structural models (A–D) ofthe C. merolae supercomplex purified from three light

Figure 6. Sections of the electron micrographs of negatively stained PSI-LHCI supercomplex particles purified from C. merolaecells grown under three different light conditions (LL, ML, and EHL).

Figure 7. Structural characterization of PSI-LHCI supercomplexes fromthe red alga C. merolae grown under different light conditions. Single-particle image analysis revealed two forms of PSI-LHCI supercomplexesunder LL conditions (A and B), four forms of PSI-LHCI supercomplexesunder ML conditions (C–F), and three forms of PSI-LHCI super-complexes under EHL conditions (G–I).


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regimes (LL,ML, and EHL), which consider the varyingnumber of Lhcr subunits, as well as the absence of somecore subunits, as determined by mass spectrometry inthis study (Table I) and in the other published data(Busch et al., 2010; Tian et al., 2017a).Structural model A corresponds to the largest form of

the PSI-LHCI supercomplex, which consists of the PSIcore complex and up to eight Lhcr antenna proteins.The inner belt of four antenna proteins is likely to beorganized within the same architecture as the higherplant LHCI structure (Mazor et al., 2017). The secondrow of antenna proteins can contain up to four addi-tional Lhcr subunits over and above the plant structure.

In structural model B, a smaller form of the PSI-LHCIsupercomplex lacks one Lhcr antenna protein in thesecond row of LHCI. Its absence can lead to differentbinding of the antenna proteins present in the inner beltof LHCI at the position where Lhca1 binds to the corecomplex in higher plants (Qin et al., 2015; Mazor et al.,2017).

Structural model C corresponds to the smaller form ofthe PSI-LHCI supercomplex, which, in addition, lacks(compared withmodel B) the PsaK subunit (Table I) andanother closely associated antenna protein. Thus, the PSIcore complex associateswith only six antenna proteins inthis type of class average.

Figure 8. Structural modeling of the PSI-LHCI supercomplex isolated from the red alga C. merolae grown under different lightconditions. The largest form of the PSI-LHCI supercomplex consists of the PSI core complex and eight antenna proteins (A);smaller forms represent the association between the PSI core complex and either seven (B andD) or six (C) Lhcr antenna subunits.Magenta contours and black asterisks indicate strong densities and stain-accumulated areas, respectively, in the projectionmap ofthe largest PSI-LHCI supercomplex (A). Overlay of the contour model with the projection maps of smaller forms of the PSI-LHCIsupercomplexes (B–D) indicates their structural differences comparedwith the largest form. Structural assignment of the PSI-LHCIsupercomplexes was based on overlay of the x-ray structure of the plant PSI-LHCI supercomplex (Mazor et al., 2017). The PsaGand PsaH subunits were removed from the structure, as they are absent in C. merolae (Matsuzaki et al., 2004). PsaK is missing inthe PSI core structure in the structural model of the smaller PSI-LHCI supercomplex (C). The structure of the Lhca4 protein wasused to fit the second row of Lhcr antenna subunits.

Table IV. Quantification of Chla/P700 ratios in LL, ML, and HL C. merolae PSI-LHCI supercomplexes

PSI-LHCI Sample Total No. of Chls No. of Chls in the LHCI Antenna Estimated No. of Lhcr Subunits

LL (35 mE m22 s21) 214.0 6 2.34 116.0 6 2.34 8ML (90 mE m22 s21) 185.6 6 4.56 87.6 6 4.56 6HL (150 mE m22 s21) 169.0 6 5.29 71.0 6 5.29 5EHL (350 mE m22 s21) 159.0 6 2.51 61.5 6 2.51 4

Each value represents an average of at least three independent measurements from two to three indepen-dent samples. The number of Lhcr subunits was estimated taking into account an average of 14 Chl mol-ecules per antenna subunit, as determined in the latest 2.6 A x-ray structure of the higher plant PSI-LHCIsupercomplex (Mazor et al., 2017).





Structural model D represents the smallest form ofthe C. merolae PSI-LHCI supercomplex. Although thePSI core complex can associate with up to seven an-tenna proteins in the same way as in model B, theprotein density close to the PsaL subunit, which is es-sential for trimerization of the cyanobacterial PSI(Chitnis and Chitnis, 1993; Schluchter et al., 1996;Jordan et al., 2001), is absent in this projection.

We compared our electron microscopy data byquantification of Lhcr subunits by measuring the P700/Chla ratios for all four PSI-LHCI complex preparationsusing chemical oxidation and reduction of the P700 RCand comparing the same measurements conducted onthe isolated PSI complex of T. elongatus, which is knownto bind 98 Chla molecules (Jordan et al., 2001). The re-sults of this analysis are summarized in Table IV. Inbrief,we estimated the total number ofChlamolecules perPSI-LHCI complex as 214, 186, 169, and 159. These valuescorrespond to eight, six, five, and four Lhcr subunits perLL, ML, HL, and EHL PSI-LHCI supercomplex, respec-tively, taking into account an average of 14 Chl moleculesper antenna subunit, as determined in the latest 2.6 Åx-ray structure of the higher plant PSI-LHCI super-complex (Mazor et al., 2017). The total number of Lhcrsubunits estimated for the smallest EHL complex usingthe redox spectroscopy approach was somewhat smallerthan in our structural model C (four versus six Lhcrs,respectively), possibly due to some deactivation of the PSIRCs exposed to EHL conditions or the experimental errorof both approaches.

Overall, our combined P700/Chla quantification andmodeling of the x-ray structure of the plant PSI-LHCIsupercomplex onto 2D projections of the C. merolaePSI-LHCI supercomplex revealed that the smallest par-ticle, which is predominant in EHL conditions, com-prises the PSI core complex, with two rows ofasymmetrically bound four to six Lhcr subunits lo-cated on the PsaF/PsaJ side of the core complex. Onthe other hand, the LL PSI-LHCI particle containedup to four additional Lhcr antenna subunits over andabove the basic unit of the PSI-LHCI complex, dis-playing the additional loosely bound protein densityextending the two rows of the proteins forming thebelt-shaped LHCI complex. Our observation of suchsignificant remodeling of the C. merolae PSI-LHCIcomplex is in stark contrast to the results of Buschet al. (2010) and Tian et al. (2017a), who reported onlythree to four Lhcr subunits forming the C. merolaeLHCI antenna domain in LL and LL/ML conditions.Nevertheless, in agreement with our study, suchsignificant structural readjustment of the LHCI an-tenna size has been observed for other microalgalspecies exposed to LL conditions, including the redalgae G. sulphuraria (Thangaraj et al., 2011) and C.caldarium (Gardian et al., 2007) as well as the greenalga Chlamydomonas reinhardtii (Drop et al., 2011).

Therefore, we propose that significant structural re-modeling of the LHCI antenna provides the molecularbasis of photoadaptation in the extremophilic red alga C.merolae, whereby, upon exposure to EHL, the LHCI

antenna has the smallest size to prevent overexcitation ofthe PSI RC. On the other hand, LL illumination induces a2-fold increase of the effective antenna size (comparedwith the smallest PSI-LHCI complex predominant in EHLand HL conditions) to maximize the solar energy capturefor efficient photochemistry to occur in the PSI RC.

CONCLUSIONS

In this work, we provided several lines of evidencethat the ultra-robust C. merolae PSI-LHCI supercomplexevokes three distinct molecular mechanisms underlyingits inherent robustness during adaptation to varyinglight conditions: (1) the accumulation of a photo-protective carotenoid Zea mainly in the LHCI antennaand, possibly, the PSI RC; (2) structural remodeling ofthe LHCI antenna and adjustment of the effective ab-sorption cross section; and (3) dynamic readjustment ofthe stoichiometry of the two PSI-LHCI isomers identifiedin the C. merolae photosynthetic apparatus accompaniedby the dissociation of the PsaK core subunit in the largerisomer or both isomers upon exposure to EHL and LLconditions, respectively. Our combined redox differencespectroscopy and single-particle analysis suggest that thelargest C. merolae PSI-LHCI supercomplex can bind up toeight Lhcr antenna subunits, which are organized as tworows on the PsaF/PsaJ side of the core complex. In con-trast to previous work (Busch et al., 2010), we found noevidence of functional coupling of the PBSs with the pu-rified C. merolae PSI-LHCI supercomplex in all four lightregimes studied, suggesting that the putative associationof PBSswith PSI is absent (Yokono et al., 2011; Ueno et al.,2017) or it is transient and may be lost during the purifi-cation procedure. Future work will address this issue.

MATERIALS AND METHODS

Cell Culturing and Isolation of Thylakoids

Cells of Cyanidioschyzon merolae strain NIES-1332 (obtained from the MicrobialCultureCollection of theNational Institute forEnvironmental Studies in Japan)werecultivated in a modified Allen medium at 42°C, pH 2.5 (Minoda et al., 2004), withcontinuous white light of 90 mE m22 s21 (ML) and bubbling with 5% CO2 in air, asdescribed in detail by Krupnik et al. (2013). Cultures were inoculated to the startOD680 of 0.05-0.07 and grownunder light intensities of 35, 90, 150, and 350mEm22 s21

(Panasonic FL40SS-ENW/37 lamps) for 6, 5, 4, and 3 d, respectively, to reach thetarget OD680 of 0.5-0.7 prior to cell harvesting and thylakoid isolation. Theprocedure of thylakoid isolation was performed as described by Krupnik et al.(2013). Final thylakoid pellets were resuspended in buffer A (40 mM MES-KOH,pH 6.1, 10mMCaCl2, 5mMMgCl2, and 25% [w/v] glycerol) at a Chla concentrationof 2 to 5 mg mL21, snap frozen in liquid N2, and stored at 280°C prior to use.

Cells of Thermosynechococcus elongatus BP-1 NIES-2133 wild-type strain (agift from M. Nowaczyk, University of Bochum) were grown in BG-11 mediumat 45°C, pH 8 (Castenholz, 1988), in continuous white light of 90 mmol photonsm22 s21 (ML) with gentle bubbling with 5%CO2 in air. Cultures were grown forapproximately 7 to 13 d to OD680 of 0.8 to 1 with continuous white light illu-mination. The procedure of thylakoid isolation was performed at 4°C in dimgreen light as described by Kuhl et al. (2000) with several modifications. Briefly,cells were harvested by centrifuging at 4,000g for 10 min, then resuspended in abuffer containing 20 mM MES-NaOH, pH 6.5, 10 mM MgCl2, and 10 mM CaCl2supplemented with a protease inhibitor cocktail (Roche), DNase I (5 mg per50 mL; Roche), and RNase I (10 mL from stock per 50 mL; Sigma-Aldrich). Thecells were centrifuged as above and resuspended in the same buffer supple-mentedwith 500mM D-mannitol and the other supplements as above. Cellswere


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disrupted byvigorous agitationwith 0.1-mmglass beads (as described byKrupniket al. [2013]) for 13 cycles, each of 10 s of beating and 4 min of rest. The cell ho-mogenate was recovered from the beads by filtering throughWhatman paper andwashing the beads with the buffer as above supplemented with 500 mM D-man-nitol. The homogenatewas centrifuged for 1min at 1,000g to remove the unbrokencells, then ultracentrifuged at 180,000g for 25 min to harvest the thylakoid mem-branes. The thylakoid pellet was resuspended in the same buffer devoid of D-mannitol. The operationwas repeated two to three times depending on the amountof PBSs present. The final thylakoid pellet was resuspended in the buffer as abovesupplemented with 500 mM D-mannitol, adjusted to a Chla concentration of 2 to5 mg mL21, snap frozen in liquid N2, and stored at 280°C prior to use.

Purification of C. merolae PSI-LHCI and T. elongatusPSI Complexes

Solubilization of C. merolae thylakoids and separation of the PSII from crudePSI-LHCI samples was performed according to the protocol described inKrupnik et al. (2013). The crude C. merolae PSI-LHCI fraction, eluted from theDEAE TOYOPEARL 650M column with 0.09 M NaCl, as described by Krupniket al. (2013), was applied onto the DEAE TOYOPEARL 650S column, andpure PSI-LHCI supercomplex was eluted with a continuous 0 to 0.2 M NaClgradient in the carrier buffer. The PSI-LHCI pool obtained after the DEAE 650Scolumn was concentrated to 1 mg mL21 Chla and further purified to removeany residual PBSs by additional chromatography purification steps performed,first, on the desalting Superdex G-25 column in buffer B (40 mM HEPES-NaOH, pH 8, 3 mM CaCl2, 25% [w/v] glycerol, and 0.03% [w/v] dodecyl-b-D-maltoside, DDM), followed by an anion-exchange chromatography (AEC)step using a UNOQ12 column. The ultrapure PSI-LHCI supercomplex fractionswere collected with a 0.05 M NaCl gradient that was separated from the PBSs,which displayed a very strong affinity to the UNOQ12 resin. The fractionscontaining the pure PSI-LHCI supercomplex were collected and concentrated to2 to 5 mg mL21 Chla, snap frozen in liquid N2, and stored at280°C prior to use.

For the purification of T. elongatus PSI trimer, thylakoids were thawed 5 h onice. Normally, a total of 66.3 mg of Chla was used for solubilization with a10% [w/v] stock of DDM (Roth). The Chla concentration was adjusted to1.61 mg mL21 (1.8 mM) before solubilization with 0.5% [w/v] DDM (10 mM) at adetergent-to-Chla molar ratio of 5:1, in the presence of the protease inhibitorcocktail (Roche), for 20min at room temperature (RT) in the dark. The solubilizedthylakoids were ultracentrifuged at 80,000g for 30 min (T-865 rotor; Thermo Sci-entific), and the supernatant was collected for the subsequent purification steps.Trimeric PSI complex of T. elongatuswas purified from solubilized thylakoids byeluting from the DEAE TOYOPEARL 650M column with 0.09 M NaCl, as de-scribed previously (Krupnik et al., 2013). The crude PSI trimerwas then applied toa DEAE TOYOPEARL 650S column that was subsequently washed extensivelywith the carrier buffer to remove excess PBSs and carotenoids. PSI trimer waseluted with a linear gradient of 0 to 0.5 M NaCl, then buffer exchanged on thedesalting SephadexG-25 column into buffer B as above, concentrated to 2 to 5mgmL21 Chla, snap frozen in liquid N2, and stored at 280°C prior to use.

The monomeric PSI complex of T. elongatus were purified according to El-Mohsnawy et al. (2010). Briefly, thylakoid membranes were resuspended with0.8 M ammonium sulfate and stirred at 50°C for 20 min in the dark. The sus-pension was cooled down to RT before the isolation of monomeric PSI complexby solubilization of the membranes with 0.6% to 1% [w/v] DDM. After ultra-centrifugation at 80,000g for 1 h at 4°C, the filtrate was subjected to two puri-fication steps hydrophobic interaction chromatography (HIC) and AEC topurify the monomeric PSI complex.

The Chla concentration was measured spectroscopically according to Porraet al. (1989) using an extinction coefficient of 86.3 mg mL21 cm21. Purity of thesamples was confirmed spectroscopically, by size exclusion chromatography,and by SDS-PAGE, as described by Krupnik et al. (2013).

Isolation and Biochemical Characterization of C. merolaeLHCI Antenna and LHCI-Depleted PSI RC Particles

TheLHCIantennaandPSI coreparticleswere isolatedbysolubilizationof thePSI-LHCI supercomplex (0.3 mg Chla mL21) with 1.5% [w/v] DDM and 0.6%[w/v] Zwittergent 3-16 and with five freeze/thaw cycles followed by Sucgradient centrifugation of the detergent-treated complexes, as described byMelkozernov et al. (2004). Fractions were collected following 17 h of ultracen-trifugation at 140,000g (Surespin 630 rotor; Thermo Scientific), concentrated to1 to 3 mg mL21 Chla, and characterized by SDS-PAGE, RT absorption, and 77Kfluorescence spectroscopy, as described below.

HPLC Pigment Analysis

Analytical HPLC of pigments was performed according to the method de-scribed byKrupnik et al. (2013) using aNucleosil-100 C18 column (Teknokoma)and a linear gradient of 10% to 60% [v/v] ethyl acetate. The content of eachpigment was expressed as a ratio of the area under the pigment-correspondingpeak to the area under the Chla peak. For the pigment molar ratio calculations,extinction coefficients of 83.2, 91.7, and 125.3 mM

21 cm21 for Zea, Chla, andb-car were used, respectively (Oren et al., 1996).

Activity of PSI

Photochemical activity of the purified PSI-LHCI (2.5 mg of Chla) was mea-sured by the oxygen consumption assay (Vernon and Cardon, 1982;Allakhverdiev et al., 2000) using an oxygen Clark-type electrode (Hansatech).Standard measurements were performed at 30°C in the reaction buffer (40 mM

HEPES-NaOH, pH 8, 3 mM CaCl2, 25% [w/v] glycerol, and 0.05% [w/v] DDM)in the presence of 0.2 mM methyl viologen as an exogenous electron acceptorwith 10 mM sodium azide as an efficient physical quencher of singlet oxygenand 0.2 mM dichlorophenolindophenol as a mediator. For a standard mea-surement, the samples were incubated in the dark for 2 min, followed by theaddition of 6 mM sodium ascorbate as the sacrificial electron donor and illu-mination with a white light intensity of 5,000 mE m22 s21, using a KL 2500 LCDwhite light source (Schott).

For measurements of the activity of PSI in the wide pH range, pH wasregulated by adding HCl or NaOH into the buffer containing 40 mM HEPES-NaOH, 3 mM CaCl2, 25% [w/v] glycerol, and 0.05% [w/v] DDM. Samples werepreincubated at various pH levels in the dark for 30min with dilution of at least50 times. The oxygen consumption was then immediately measured in the re-action buffer: 40 mMHEPES-NaOH, pH 8, 3mMCaCl2, 25% [w/v] glycerol, and0.05% [w/v] DDM. Activity was assayed with at least three independentmeasurements, and the values were expressed as means 6 SD.

RT Absorption and Circular Dichroism Spectroscopy

Optical absorption spectra were recorded at 5 mg Chla mL21 at RT in therange 800 to 350 nm using a UV-1800 Shimadzu spectrophotometer with theTCC-100 temperature-controlled cell holder, using a quartz cuvette with anoptical path length of 10 mm. Determination of Chla/P700 ratios was done bydifferential absorption spectroscopy, as described (Kargul et al., 2003). Thesamples were diluted to 20 mgmL21 in 50mMMES-KOH, pH 6.5, 10 mMMgCl2,10 mM CaCl2, and 0.05% [w/v] DDM. Chla/P700 ratios were quantified bymeasuring absorbance changes at 700 nm in the presence of 2 mM sodium as-corbate, 0.2 mM dichlorophenolindophenol as a reductant, and 0.5 mM ferri-cyanide as an oxidant.

Circular dichroism (CD) absorption spectra of PSI complexesweremeasuredaccording to Schlodder et al. (2007). PSI complexeswere diluted to 10mMChla in20 mM MES-KOH, pH 6.5, 10 mM CaCl2, 30 mM MgCl2, and 0.02% [w/v] DDM.A JASCO J-715 spectropolarimeter was adjusted to a speed of 20 nm min21,bandwidth of 2 nm, and pitch of 0.2 nm. After five cycles, the spectra wereaveraged using JASCO software.

77K Fluorescence Spectroscopy

Steady-state fluorescence emission spectra at 77K were acquired in an LS55Fluorescence Spectrometer (Perkin Elmer), as described (Busch et al., 2010;Krupnik et al., 2013). For spectroscopic measurements, PSI samples (3 mg ofChla) were diluted in buffer B, then diluted 2-fold with 80% [w/v] glycerol andfrozen in liquid N2. Fluorescence emission spectra were recorded in the range600 to 800 nm using excitation wavelengths of 435 and 600 nm for Chla andphycocyanin, respectively. The emission spectra were normalized to the Chlpeak of PSI at 728 nm. Action spectra were generated by exciting the samples inthe range 400 to 700 nm and recording emission at 728 nm. The spectra obtainedwere normalized to the Chl peak at 674.5 nm. All spectra were obtained fromtwo to three independent preparations in three replicates.

BN-PAGE

BN gel electrophoresis experiments were performed with 3% to 12% non-denaturing continuous gradient polyacrylamide gels according to Schägger andvon Jagow (1991). Samples of PSI at 0.5 mg Chl mL21 were mixed with 0.25volumes of Coomassie Blue solution (5% [w/v] Serva Blue G, 750 mM





aminocaproic acid, and 35% [w/v] Suc), and after incubation, they were loadedonto the gel. Commercially available markers from Invitrogen were used forprotein mass identification. Electrophoresis was conducted in the runningbuffer at 90 V for 15 h at 4°C in the dark according to Farci et al. (2017).

Densitometry analysis of the intensity of the bands on the BN gel was per-formed using Image Lab software (Bio-Rad Molecular Imager GelDoc XR).

Sample Preparation for Mass Spectrometry

The bands from the BN gels were excised, reduced in DTT (10mM, 56°C, and30 min), and subsequently alkylated with iodoacetamide (55 mM, 25°C, and20 min in the dark). Following dehydration with acetonitrile, trypsin (1 ng mL21

solution in 50 mM ammonium bicarbonate) was added, and the gel pieces wereallowed to swell on ice for 30 min. They were then digested overnight at 37°Cwith shaking. After digestion, the peptide content was extracted twice withsonication (using a solution of 50:50 water:acetonitrile and 1% [v/v] formicacid). The pooled extracts were placed in a clean tube and dried with a speedvacuum centrifuge. The dried pool was finally redissolved in 50 mL of OasisSolvent A (water and 0.05% [v/v] formic acid). The digests were then desaltedwith the Waters Oasis HLB mElution Plate 30 mm in the presence of a slowvacuum. In this process, the columnswere conditionedwith 33 100mL of OasisSolvent B (80% acetonitrile [v/v] and 0.05% [v/v] formic acid) and equilibratedwith 3 3 100 mL of Oasis Solvent A. The samples were loaded, washed threetimes with 100 mL of Oasis Solvent A, and then eluted into PCR tubes with50 mL of Oasis Solvent B. The eluates were dried down with the speed vacuumcentrifuge and dissolved in 20 mL of 5% [v/v] acetonitrile, 95% MilliQ water,and 0.1% [v/v] formic acid prior to analysis by liquid chromatography-tandemmass spectrometry (LC-MS/MS).

Liquid Chromatography-MS/MS Analysis

Peptides were separated using the nanoAcquity ultra-performance liquidchromatography system (Waters) fitted with a trapping column (nanoAcquitySymmetry C18, 5mm, 180mm3 20mm) and an analytical column (nanoAcquityBEH C18, 1.7 mm, 75 mm 3 250 mm). The outlet of the analytical column wascoupled directly to an Orbitrap Fusion Lumos (Thermo Fisher Scientific) usingthe Proxeon nanospray source. Solvent Awas water and 0.1% [v/v] formic acidand solvent B was acetonitrile and 0.1% [v/v] formic acid. The samples (5 mL)were loaded with a constant flow of solvent A (5 mL min21) onto the trappingcolumn. Trapping time was 6 min. Peptides were eluted via the analyticalcolumn with constant flow (0.3 mL min21). During the elution step, the per-centage of solvent B increased in a linear fashion from 3% to 25% in 30min, thenincreased to 32% in 5min, and finally to 50% in a further 0.1 min. Total run timewas 60 min. The peptides were introduced into the mass spectrometer via aPico-Tip Emitter 360-mm o.d. 3 20-mm i.d., 10-mm tip (New Objective), and aspray voltage of 2.2 kVwas applied. The capillary temperaturewas set at 300°C.The ion funnel radio frequency (RF) lens was set to 30%. Full-scan mass spectrawith mass range 375 to 1,500 mass-to-charge ratio (m/z) were acquired in profilemode in the Orbitrap with resolution of 120,000. The filling time was set at amaximum of 50 ms with limitation of 2 3 105 ions. The top speed method wasemployed to take the maximum number of precursor ions (with an intensitythreshold of 5 3 103) from the full-scan mass spectra for fragmentation (usinghigher-energy collisional dissociation orHCD of 30%) and quadrupole isolation(1.4-D window) and measurement in the ion trap, with a cycle time of 3 s. Themonoisotopic precursor selection peptide algorithm was employed, but withrelaxed restrictions when too few precursors meeting the criteria were found.The fragmentationwas performed after the accumulation of 23 103 ions or afterfilling time of 300 ms for each precursor ion (whichever occurred first). MS/MSdata were acquired in centroid mode, with the rapid scan rate and a fixed firstmass of 120m/z. Onlymultiply charged (2+ to 7+) precursor ions were selected forMS/MS. Dynamic exclusion was employed with a maximum retention period of60 s and relative mass window of 10 ppm. Isotopes were excluded. Additionally,only one data-dependent scan was performed per precursor (only the most in-tense charge state was selected). Ions were injected for all available parallelizabletime. In order to improve the mass accuracy, a lock mass correction using abackground ion (m/z 445.12003) was applied. For data acquisition and processingof the raw data, Xcalibur 4.0 (Thermo Scientific) was employed.

Proteomic Data Analysis

Raw data from the mass spectra were searched using MaxQuant (version1.5.3.30; Cox and Mann, 2008). Data were searched against a species-specific

(C. merolae; http://merolae.biol.s.u-tokyo.ac.jp/download/cds.fasta) data-base, with a list of common contaminants appended using the Andromedasearch engine (Cox et al., 2011). The search criteria were set as follows: fulltryptic specificity was required (cleavage after Lys or Arg residues, unlessfollowed by Pro); two missed cleavages were allowed; oxidation (M) andacetylation (protein N-term) were applied as variable modifications, carbami-domethyl Cys was applied as a fixed modification, and a mass tolerance of20 ppm (precursor) and 0.5 D (fragments) was set. The reversed sequences ofthe target database were used as a decoy database. Peptide and protein hitswere filtered at a false discovery rate of 1% using a target-decoy strategy (Eliasand Gygi, 2007). Data from entries that are relevant to the photosynthetic ap-paratus are represented in Table I. They are depicted according to their iBAQintensities (extracted from theMaxQuant protein groups output) for each of theproteins corresponding to each of the BN complexes from the BN-PAGE gels(Fig. 3). Here, the relative contributions are represented by icons, where fourcrosses represent proteins with iBAQ values $ 1.75e10, three crosses $ 2.5e9,two crosses $ 5e8, one cross $ 2.5e6, and a minus sign , 2.5e6. For a full listof proteins identified in each band (including those not considered to be partof the photosynthetic apparatus), with absolute iBAQ values shown, seeSupplemental Table S1. Datawere filtered according to iBAQvalue; any proteinwith an iBAQ value less than 1e6 in any band was filtered from the list. Onepeptide per protein hits were retained where the iBAQ value was above thisthreshold in at least one condition, as a number of the proteins with this peptidenumber belong to the lower molecular weight proteins in the list, where not somany peptides can be expected upon digestion.

Electron Microscopy and Image Processing

Specimens for electron microscopy were prepared on glow-dischargedcarbon-coated copper grids and negatively stained with 2% [w/v] uranyl ac-etate. Electron microscopy was performed on a Tecnai TF20 microscope (FEI)equipped with a field emission gun operated at 200 kV. Images were recordedwith an Eagle 4KCCD camera (FEI) at 83,0003magnificationwith a pixel size of0.18 nm. Automated data acquisition software for single-particle analysis (EPU;FEI)was used for the acquisition of about 800, 1,100, and 700micrographs of LL,ML, and HL samples, respectively. Data sets of ?60,000, 150,000, and 90,000single-particle projections of PSI-LHCI supercomplexes were selected for LL,ML, and HL samples, respectively. Single-particle image analysis (Boekemaet al., 2009) was performed using RELION software (Scheres, 2012). Imageanalysis revealed that about 75% to 80% of the projections from each data setcould be assigned to one of the specific classes of the PSI-LHCI supercomplexes.Pseudo-atomic models of the PSI-LHCI supercomplexes were created usingPYMOL (DeLano, 2002).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBLdata libraries under accession numbers. CMV135C, CMV136C, CMV059C,CMV144C, CMV128C, CMV201C, CMV202C, CMV055C, CMV236C,CMP086C, CMN234C, CMN235C, CMQ142C, CMV063C, CMP166C,CMV158C, CMV064C, CMV159C, and CMV051C.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. HPLC pigment analysis of the C. merolae PSI-LHCI supercomplex.

Supplemental Figure S2. HPLC pigment analysis of the C. merolae cells.

Supplemental Figure S3. CD-absorption spectra of PSI monomers andtrimers of T. elongatus and the ML PSI-LHCI supercomplex of C. merolae.

Supplemental Table S1. Full list of proteins identified by mass spectrom-etry in each BN-PAGE band visualized in Figure 3.

ACKNOWLEDGMENTS

We thank Dr. Marc Nowaczyk (University of Bochum) for providing theT. elongatus wild-type strain BP-1 NIES 2133.

Received July 25, 2017; accepted November 28, 2017; November 29, 2017.


Haniewicz et al.


http://merolae.biol.s.u-tokyo.ac.jp/download/cds.fasta







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Molecular Mechanisms of Photoadaptation of Photosystem I ... · Dissecting the molecular mechanisms...

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