Temperature-Induced Remodeling of the Photosynthetic ...tolerant photosynthetic eukaryotes (Ciniglia et al., 2004; Kobayashi et al., 2014) their main competitors in these hostile environments

Temperature-Induced Remodeling of the PhotosyntheticMachinery Tunes Photosynthesis in the ThermophilicAlga Cyanidioschyzon merolae1

Denitsa Nikolova2, Dieter Weber2,3, Martin Scholz, Till Bald, Jörn Peter Scharsack, and Michael Hippler*

Institute of Plant Biology and Biotechnology (D.N., D.W., M.S., T.B., M.H.), Institute for Evolution andBiodiversity (J.P.S.), University of Münster, 48143 Münster, Germany

ORCID IDs: 0000-0001-5341-0079 (T.B.); 0000-0003-4291-6853 (J.P.S.); 0000-0001-9670-6101 (M.H.).

The thermophilic alga C. merolae thrives in extreme environments (low pH and temperature between 40°C and 56°C). In thisstudy, we investigated the acclimation process of the alga to a colder temperature (25°C). A long-term cell growth experimentrevealed an extensive remodeling of the photosynthetic apparatus in the first 250 h of acclimation, which was followed by cellgrowth to an even higher density than the control (grown at 42°C) cell density. Once the cells were shifted to the lowertemperature, the proteins of the light-harvesting antenna were greatly down-regulated and the phycobilisome compositionwas altered. The amount of PSI and PSII subunits was also decreased, but the chlorophyll to photosystems ratio remainedunchanged. The 25°C cells possessed a less efficient photon-to-oxygen conversion rate and require a 2.5 times higher lightintensity to reach maximum photosynthetic efficiency. With respect to chlorophyll, however, the photosynthetic oxygenevolution rate of the 25°C culture was 2 times higher than the control. Quantitative proteomics revealed that acclimationrequires, besides remodeling of the photosynthetic apparatus, also adjustment of the machinery for protein folding,degradation, and homeostasis. In summary, these remodeling processes tuned photosynthesis according to the demandsplaced on the system and revealed the capability of C. merolae to grow under a broad range of temperatures.

Extremophilic organisms like the members of therhodophytan order Cyanidiales occupy harsh ecologi-cal niches, being able to withstand very low pH con-ditions (pH 0.5–3) and relatively high temperatures(40°C–56°C). Phylogenetic studies indicate that they arethe most ancient clade of red algae having diverged atthe base of the Rhodophyta about 1.3 billion years ago(Yoon et al., 2004, 2006). Considered the most heat-tolerant photosynthetic eukaryotes (Ciniglia et al.,2004; Kobayashi et al., 2014) their main competitors inthese hostile environments are mostly prokaryotes.However, with varying ecological conditions, Cyani-diales are able to persist at temperatures as low as ap-proximately 35°C in aquatic sites or 10°C in soil,

because below these temperature limits, there is toomuch competitionwith other acidophilic algae (Doemeland Brock, 1971; Reeb and Bhattacharya, 2010).

Cyanidioschyzon merolae, one of the two Cyanidialeswith a fully sequenced genome (Ohta et al., 2003;Matsuzaki et al., 2004; Nozaki et al., 2007), is an im-portant eukaryotic model organism because of itsunique genomic and proteomic characteristics. With its16.5 Mbp, the genome of C. merolae has one of thesmallest sizes for a photosynthetic eukaryote. Further-more, it has a high degree of gene compaction since itpossesses only 0.5% intron-containing protein genesand low gene redundancy (Ohta et al., 2003; Matsuzakiet al., 2004). This and the very small proteome allowstudies on the origin, evolution, primary, and second-ary endosymbiosis as well as complex fundamentalprocesses of eukaryotic cells (Matsuzaki et al., 2004).Furthermore, the C. merolae cell has the simplest struc-ture among all photosynthetic eukaryotes with no cellwall and no vacuole, one nucleus, one mitochondrion,and one chloroplast as well as a reduced set of otherultrastructural components (Merola et al., 1981;Kuroiwa et al., 1994).

C. merolae is intriguing also regarding the evolutionof the photosynthetic machinery. In prokaryotic cya-nobacteria, the antenna system consists of solely chlo-rophyll a and phycobilisomes (PBSs), which were lostmultiple times in the course of eukaryotic evolutionand replaced by thylakoid membrane-integral light-harvesting complexes (LHCs) in green algae andhigher plants (Neilson andDurnford, 2010). Containing

1 This work was supported by the Deutsche Forschungsgemein-schaft (HI 739/13-1 to M.H.).

2 These authors contributed equally to the article.3 Present address: Institute of Applied Microbiology, Aachen Biol-

ogy and Biotechnology, RWTH Aachen University, 52074 Aachen,Germany.

* 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:Michael Hippler ([email protected]).

D.W. and M.H. designed the research; D.W. and D.N performedthe research; J.P.S. provided technical assistance to D.N. and D.W.;D.N., D.W., M.S., and M.H. analyzed the data; T.B. developed scriptsfor analysis of proteomics data; D.N., D.W., and M.H. wrote themanuscript.

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the unique combination of both antenna types, Rho-dophyta represents an important evolutionary inter-mediate. Besides phycobilisomes the members of thegroup encode a species-dependent number of chloro-phyll a-binding LHCs, which functionally associatesolely with PSI (Wolfe et al., 1994; Busch et al., 2010).

Phycobilisomes greatly expand the solar spectrumused for photosynthesis, because they absorb in thespectral gap between the blue and main-red absorptionof chlorophyll. Located on the outer surface of thethylakoid membrane, the PBSs are assembled fromvarious types of phycobiliproteins, which have cova-lently bound light-absorbing tetrapyrrole chromo-phores, and from nonpigmented linker polypeptides,which are involved in the assembly and stability of thecomplex at different levels, but also facilitate efficientflow of excitation energy. Four major subgroups ofphycobiliproteins have been described: allophycocya-nin, at the membrane surface, forms the core structureof the PBS, whereas the series of rods radiating out fromthe core is composed of phycocyanin (closest to thecore), and, distal to the core, phycoerythrin and phy-coerythrocyanin (latter one is absent in red algae; Adir,2005). Furthermore, C. merolae and the closely relatedred alga Galdieria sulphuraria also lack phycoerithrin(responsible for red coloring); thus, the algae appearblue-green in color (Stadnichuk et al., 2011). Light en-ergy is absorbed by the distal end of the rods andtransferred toward allophycocyanin, which can supplyexcitation to the chlorophyll pigments in the photo-synthetic reaction centers. The basic core-rod structureis widely conserved among cyanobacteria and algae,but there is a great diversity across different speciesregarding phycobiliproteins, core structure type, andlinker polypeptides (rod linkers and rod-core linkers).The diversity of the latter is important for the structuraldiversity of peripheral rods (Kondo et al., 2007).

Although PBSs are primarily attached to PSII, thepresence of functional, specific phycobilisome PSI an-tenna in cyanobacteria and C. merolae was demon-strated. Kondo et al. (2007) identified two types of PBSin the cyanobacterium Synechocystis sp. PCC 6803, dif-fering in their rod-core linkers (CpcG1- and CpcG2-PBS) and demonstrated a 3-fold higher efficiency fromCpcG2-PBS to PSI than to PSII. Busch et al. (2010) alsoshowed a PSI-specific phycobilisome subcomplex inC. merolae, which is able to transfer excitation energy toPSI. It lacks allophycocyanin and contains phycocyaninand a CpcG-like rod-core linker polypeptide.

Recently, a megacomplex containing PBS, PSII, andPSI was isolated from the cyanobacterium SynechocystisPCC 6803 after in vivo protein cross linking, demon-strating that PBS can supply excitation energy to bothphotosystems (Liu et al., 2013). In the megacomplex,four PSII subunits (PsbB, PsbC, PsbD, PsbI) are linkedto the phycobilisome linker polypeptide ApcE, whereasApcD is located on the edge area of PSI through a coveformed by PsaD and PsaA (Liu et al., 2013). Duringdark-light transitions, however, such PBS-PSII-PSImegacomplexes are not active in vivo, but modulation

of the excitation energy transfer to the photosystems isregulated by functional uncoupling of PBS from thePSI and almost no reattachment to PSII (Chukhutsinaet al., 2015).

Since the mobility of the most transmembrane pro-teins in the thylakoid membrane is very restricted,mobility of the extrinsic antenna is thought to play animportant role in photoprotection in cyanobacteria andmesophilic red algae by the process of state transition(Kaňa et al., 2014; Kirilovsky, 2015). In thermophilicalgae, however, excess light is dissipated via the pro-cess of nonphotochemical quenching as the mainphotoprotective mechanism (Krupnik et al., 2013).The extremophilic alga C. caldarium exhibits highly re-stricted phycobilisome mobility, which is due to thestrength of the PBS-photosystem interaction rather thanto macromolecular crowding and a decrease in lipiddesaturation (Kaňa et al., 2014). The latter is consideredto be key in acclimation process to lower temperaturesin mesophilic and to some extent in thermophilic cya-nobacteria (Kiseleva et al., 1999; Murata and Los, 1997).However, increased PBS mobility in the thermophilicalga Cyanidium caldarium could be achieved by lower-ing the growth temperatures, which leads to weakerPBS-photosystem binding (Kaňa et al., 2014).

In this study, we investigated the acclimation of thethermophilic alga C. merolae to lower temperatures(25°C versus 42°C). Interestingly, our data revealed thatC. merolae is well capable of growing at 25°C. Growth atthis temperature, however, is accompanied by a sub-stantial remodeling of the photosynthetic machinery.

RESULTS

Hereinwe investigated the ability of the thermophilicalga C. merolae to acclimate to colder temperatures.

Under Temperature Stress, the Alga Started Growing Oncethe Remodeling of the Photosynthetic Machinery WasCompleted and Outgrew the Control

To monitor and compare the growth behavior of thealga, two cultures emanating from a 42°C control cul-ture were grown at 25°C and 42°C, respectively. Celldensity and the fluorescence of cyanobilin and chloro-phyll were recorded via flow cytometry in two inde-pendent experiments (Fig. 1; Supplemental Fig. S1).For further evaluation, only the data of living cells(4,6-diamidino-2-phenylindole [DAPI] negative) wereconsidered. Shifting of cells to a colder temperatureresulted in an immediate and steady decrease ofthe cyanobilin and chlorophyll fluorescence (Fig. 1;Supplemental Fig. S1). However, low fluorescence in-tensities remained almost unchanged in the course ofthe experiment. Cells cultivated at 42°C on the contraryshowed an overall continuous increase in pigmentfluorescence.

After an approximately 250 h of temperature accli-mation, the 25°C cells started growing with a growth

36 Plant Physiol. Vol. 174, 2017

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rate comparable to the control and reached a 2- to 3-foldhigher cell density (Fig. 1; Supplemental Fig. S1).Quantitative proteomics on whole cells revealed

down-regulation of antenna proteins, PSI, and PSIIsubunits.For detailed studies of the acclimation process,

quantitative proteomics was employed. Cells grownat 42°C were metabolically labeled with heavy nitro-gen, whereas the 25°C cells were grown in normal 14N-labeled medium. Three different protein sets wereanalyzed: a mix of 42°C and 25°C protein extract (twoindependent biological replicates, designated set1 andset3) and a protein mix of 42°C and 25°C long-termadapted culture (designated set2; see “Materials andMethods”).As indicated by the pigmentfluorescence in the growth

monitoring experiment, all identified light-harvestingantenna proteins were down-regulated at 25°C com-pared to the control (Fig. 2C; Supplemental Table S1). ThePBS antenna proteins (phycocyanin, allophycocyanin,and the linker polypeptides) were much more affected(down to 2%–10%) than the chlorophyll containing LHCproteins (CMN235C, CMQ142C, down to 10%–20%).Furthermore, phycocyanin-binding proteins were stron-ger down-regulated than allophycocyanin-binding pro-teins, as was the phycocyanin-associated rod linkerprotein (CMP166C) compared to the phycobilisomelinker (CMV157C, ApcE) and rod-core linker (CMV051C,CpcG) polypeptides (Fig. 2C). The fact that the differentchains of phyco- and allophycocyanin-binding proteinswere quantified with similar ratios among each other

strongly supports the data. All of these identified phy-cobilisome antenna proteins were among the top20 down-regulated proteins (Fig. 2G).

Additionally, most of the photosystem subunits weredown-regulated at the lower temperature (Fig. 2D;Supplemental Table S1). The subunits of the ATP syn-thase, FNR as well as ferredoxin and components of thecytochrome b6f complexwere less affected compared tothe PSI and PSII units (Fig. 2D; Supplemental Table S1).Four of the PSII subunits (D1 and D2 core proteins aswell as PsbC and PsbO) are among the top 20 down-regulated proteins (Fig. 2G). Furthermore, proteinsinvolved in the stability and biogenesis of PSII werealso slightly down-regulated (Fig. 2D; SupplementalTable S1).

The majority of the proteins involved in differentcellular respiration processes were similarly expressedin both conditions (Fig. 2E; Supplemental Table S1). Theenzymes controlling the first phase of glycolysis werestronger affected by the lower temperature (2-folddown-regulation) than the ones of the last two steps ofthe pathway. The identified proteins of the Krebs cyclewere differently regulated. The identified subunits ofthe mitochondrial ATP synthase showed no change inabundance at 25°C.

Most of the identified stress-related proteins like pro-teins responsible for proper protein folding (chaperonesand chaperonins, heat-shock proteins [proteins of both oftheHsp70 andHsp90 families]), quality control (calnexin),and degradation of wrongly folded proteins (ubiquitin-carboxyl-terminal hydrolase, ubiquitinyl hydrolase 1,ubiquitin-activating enzyme) were up-regulated in cellsgrown at 25°C (Fig. 2F; Supplemental Table S1). HSP90,ubiquitin-activating enzyme and chloroplast chaperoninCPN60 were furthermore among the top 20 up-regulatedproteins (Fig. 2H). Interestingly, whereas a protein similarto thioredoxin h was up-regulated (Supplemental TableS1), proteins directly involved in reactive oxygen species(ROS) defense (catalase and superoxide dismutase) weredown at least by 50%.

The most affected protein by the low temperature inall three sets was the phycocyanin-associated rod linkerprotein CMP166C (25°C/42°C ratio: 0.029 6 0.01).Furthermore, besides all identified antenna proteinsand four PSII and two PSI subunits, among the top20 down-regulated proteins were also the steroidmono-oxygenase (CML339C), the Mn superoxide dis-mutase (CMT028C) and three hypothetical proteins(CMP346C, CMJ121C, and CMH239C; Fig. 2G). Al-though quantified in two of the three sets among thetop 20 down-regulated proteins is also the CpcGPBS rod-core linker polypeptide with a 23-fold down-regulation (Supplemental Table S1).

Among the most up-regulated proteins were, besidesmany stress-related proteins, also proteins involvedin nucleic acid binding and the modification ofnucleic acid structures (CMM134C uncharacterizedprotein, polyadenylate-binding protein, RNA helicase,DNA gyrase, and exodeoxyribonuclease; Fig. 2H;Supplemental Table S1). Furthermore, the translational

Figure 1. Growth and pigment fluorescence monitoring of C. merolae25°C and 42°C cultures (first biological replicate). Shown is the averageof 1 mL triplicates of each culture, which were measured every 72 h viaflow cytometry. DAPI was employed for discrimination of dead cells.Cell density of DAPI-negative cells (designated to be alive) and themean pigment fluorescence signal per cell are shown. Chlorophyll wasexcited with a 488 nm laser beam; the signal was detected at 695 nm.Phycocyanin was excited with a 633 nm laser beam, and the signal wasdetected at 660 nm.

Plant Physiol. Vol. 174, 2017 37

Remodeling of Photosynthetic Machinery in C. merolae















Figure 2. Immunoblot and comparative quantitative proteomic analysis of C. merolae whole cells grown at 25°C or 42°C. A,Immunoblot analysis of 30 mg total protein extract samples reveals the severe down-regulation of phycocyanin a- and b-chain(antiphycocyanin antibody) in the culture grown at suboptimal temperature. ATPase subunit b (ATPB) served as loading control. B,


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elongation factor eIF-4G was found to be highlyup-regulated, as were enzymes involved in the aminoacid biosynthesis (aspartokinase, vitamin B12 inde-pendent Met synthase, adenosylhomocysteinase) andcatabolism (2-amino-3-ketobutyrate coenzymeA ligase).Although quantified in one of the three sets highlyup-regulated at 25°C was the Mg-protoporphyrin IX

chelatase, which is involved in chlorophyll biosynthesis(Supplemental Table S1).

C. merolae Cells Acclimated at 25°C Possess a Less-EfficientPhoton-to-Oxygen Conversion Rate

Cells from both conditions were collected from theexponential growth phase and subjected to an oxygenelectrode. Cells were dark incubated for 20min either at25°C or 42°C before each measurement, which wascarried out at 42°C in the dark in order to measure O2consumption or at light intensities from 50 to 500 mmolphotons m22 s21 to record O2 evolution. When equal-ized on the same cell number, the 42°C culture exhibitstwo to three times higher oxygen evolution dependingon the light intensity (Fig. 3B) and very similar respi-ration capacity. With respect to chlorophyll, however,the photosynthetic oxygen evolution rate of the 25°Cculture was two times higher compared to the control(Fig. 3A), even though the amount of chlorophyll andchlorophyll containing LHC proteins was strongly di-minished (Fig. 2C).

Furthermore, the control culture reached its maxi-mum photosynthetic efficiency at lower light intensities,since its O2 evolution was already saturated at around100 mmol photons m22 s21. The 25°C culture howevershowed a steady rise of the produced oxygen with in-creasing light intensities, which flattens out at 250 mmolphotons m22 s21, corresponding to a 2.5 times higherlight intensity that is needed to achieve maximum pho-tosynthetic efficiency. Notably, this finding was similarto one observed in Synechocystis sp PCC 6803 strainlacking a PBS linker polypeptide (Shen et al., 1993).

Spectroscopic and Mass Spectrometric Analysis onIsolated Thylakoids

To investigate the acclimation of the photosyntheticapparatus of C. merolae, isolated thylakoid membraneswere solubilized with detergent and fractionated viaSuc density centrifugation (Fig. 4). Each fraction wasanalyzed by fluorescence spectroscopy (Fig. 5) andadditionally digestedwith trypsin according to the filter-aided sample preparation (FASP) protocol (Wi�sniewskiet al., 2009). Peptides were then examined by liquidchromatography-tandemmass spectrometry (LC-MS/MS;Fig. 6).

Changes of the pigment composition caused by thelower temperature were directly visible on the Suc

Figure 2. (Continued.)Key to heat map. The heat map represents the log2 ratios of the protein ranges, from black to green indicating an up-regulation,and fromblack to red indicating a down-regulation. The SD is visualized by the size of the box; the smaller the box the higher the SD

of the protein ratio. C to H, Whole-cell extracts of exponentially growing 25°C versus 15N-labeled 42°C cultures were mixed onequal protein amount (50 mg) and separated by SDS-PAGE. The resulting bands were analyzed by LC-MS/MS. Three differentprotein sets were analyzed: 42°C culture mixed with 25°C culture switched from 42°C (1), a second biological replicate of thesame conditions (3), and 42°C culture mixed with long-term adapted 25°C culture (2). Quantification was performed by qTrace(Terashima et al., 2010) after identification by OMSSA (Geer et al., 2004) and X! Tandem.

Figure 3. Oxygen evolution and consumption rates of C. merolae cellsgrown at 25°C and 42°C and measured at light intensities from 50 to500 mmol photons m22 s21. Exponentially growing cells were dark in-cubated for 20 min at the corresponding temperature before eachmeasurement, which was performed using a Clark-type oxygen elec-trode at 42°C at different light intensities. Values aremeans6 SD of threeindependent biological replicates. A, Oxygen evolution and con-sumption per mg chlorophyll. B, Oxygen evolution and consumptionper 109 cells.






gradients (Fig. 4). The intact phycobilisomes as largeantenna complexes migrated to the high-density regionof the gradient and appeared blue due to the phycobi-lins. Visual comparison of both gradients indicated thatthe PBS fraction was strongly diminished in 25°C cells.

Furthermore, the fractions containing PSI, PSII, andPBS were excited at 435 nm for chlorophyll a (Fig. 5, Aand C) or at 600 nm for phycocyanin-dependent exci-tation energy transfer (Fig. 5, B andD). The fluorescenceemission was recorded from 600 to 800 nm or 620 to800 nm, respectively, and all spectra were normalizedto the spectrum baseline at 600 nm or 620 nm.

When the 42°C phycobilisome fraction (fraction 13)was excited at 600 nm (Fig. 5B) an efficient energytransfer within intact PBS was indicated by the lowphyco- (642 nm) and allophycocyanin (655 nm) fluo-rescence emission and the strong 681nm peak, origi-nating from terminal energy acceptor ApcE (Buschet al., 2010; Su et al., 1992; Shen et al., 1993), whichstabilizes the phycobilisome core architecture. In the25°C culture the ApcE fluorescence emission wasgreatly reduced (Fig. 5D). The presence of a PSI emis-sion peak, after excitation of chlorophyll a molecules,indicated remaining PSI in the phycobilisome fraction(Fig. 5C).

With decreasing Suc density within the gradient, theemission spectra of the fractions revealed a dominating

emission peak at 728 nm (fractions 18–21) when ex-cited at 435 nm (Fig. 5, A and C), reflecting the fluo-rescence of red chlorophylls from PSI (Bruce et al.,1985; Busch et al., 2010; Kaňa et al., 2014). On theother hand, in fractions 21 to 23 the strong emissionpeaks at 686 nm and 692 nm suggested the presenceof PSII. Thus, fractions 21 to 23 (and to some extentfraction 20 of the 25°C sample) contained both PSIand PSII.

When excited at 600 nm (Fig. 5, B and D) the PSIenriched fractions (fractions 18–21) exhibited twophycobilin peaks at 642 nm and at 655 nm as well as thePSI peak at 728 nm, indicating that antenna proteinsmigrated with PSI at the same Suc density. This comi-gration was strongly diminished, when thylakoidswere treated with NaBr prior to separation on the Sucgradients (Supplemental Fig. S2, D and H). For the42°C as well as the 25°C sample, this led to a 2-foldreduction of the 642 nm and 655 nm peaks in the PSIfractions (Supplemental Figure S2, B, D, F, and H),whereas the fluorescence of the PSII antenna de-creased around four times in the 42°C and 2 times inthe 25°C culture.

MS/MS analysis (Fig. 6) of the Suc fractions con-firmed the spectroscopic data about the distribution ofthe protein complexes within the gradient and allowedthe comparison of both cultures. In contrast to the massspectrometric analysis of whole cells, where equalprotein amount of both samples was employed, for thisexperiment, the samples were equalized to the samechlorophyll concentration prior to the sucrose densitygradient (SDG) ultracentrifugation. For all further ex-periments (Figs. 5 and 6; Supplemental Figs S2 and S3),same volume hence same chlorophyll amount of thecorresponding SDG fractions was employed. Thus, al-though the 25°C whole cell exhibited reduced PSI, PSII,and chlorophyll content (Figs. 1 and 2D) as well aslight-harvesting proteins (Fig. 2C), when loading thesame Chl amount on the gradient, PSI and PSII con-centration was comparable in both conditions (Fig. 6).Furthermore, both photosystems peaked in differentfractions: PSI was concentrated in fractions 18 to 22,whereas PSII in fractions 20 to 24 (Fig. 6). Light-harvesting proteins peaked similarly to PSI, whichis even clearer in the second biological replicate(Supplemental Fig. S3).

With respect to phyco- and allophycocyanin, a 2- to4-fold down-regulationwas present in the PBS fractions(fractions 10 to 15 of the SDGs) of the 25°C samplecompared to the control, which was consistent with thewhole-cell results. The sum of the protein intensity of allallophycocyanin subunits that migrated together withPSI and PSII, however, did not exhibit any significantchange at the lower temperature (Fig. 6; SupplementalFig. S3).

Phycocyanin appeared to be more strongly impactedby the low temperature, which is more pronounced inthe first biological replicate (Fig. 6) than the second(Supplemental Fig. S3). Furthermore, the impact wasgreater in the PSII fractions 22 and 23, as well as in

Figure 4. SDGs of thylakoid membranes from C. merolae 25°C and42°C cultures. Two cultures, emanating from the same 42°C culture,were grown at either 25°C or 42°C. Isolated thylakoid membranes ofboth samples, equalized on the same chlorophyll level, were solubi-lized with 0.9% b-DM and loaded on separate discontinuous SDGswith Suc concentrations from 1.3 to 0.1 M, and after ultracentrifugationthey were fractionated from the bottom to the top.


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fractions 24 to 26, where probably free proteins mi-grated, including free phycocyanin, since these fractionsexhibited strong peak at 642 nm,when phycocyaninwasexcited (data not shown).Strong reduction of the linkers was observed in every

biological and technical replicate in the 25°C sam-ple (Fig. 6; Supplemental Fig. S3). Very stronglydown-regulated was the phycocyanin-associated rodlinker protein CMP166C, which in the control samplepeaked in the low-density fractions 20 to 24 (Fig. 6;Supplemental Fig. S3). Less affected was the phycobi-lisome rod-core linker polypeptide CpcG, which hasbeen discussed to be a part of the PSI-specific antenna(Busch et al., 2010). Consistently, CpcG rather comi-grated with the PSI complex and with the free phyco-biliprotein complex, where it was more diminished at25°C as compared to its abundance in the PSI-relatedfractions.

DISCUSSION

The thermophilic red alga C. merolae has the capacityand flexibility to acclimate to a temperature of 25°C. Thisacclimation requires a pronounced remodeling of thephotosynthetic machinery permitting growth to evenhigher cell densities. A first step toward this remodelingis a pronounced degradation of the phycobilisomes (Fig.1; Supplemental Fig. S1) as revealed by the immediateand steady decrease of the phycobilisomes-associatedpigment fluorescence and further confirmed by immu-noblot analysis and mass spectrometry of whole cells(Fig. 2, A–H).

Once the photosynthetic machinery was remodeled(after approximately 250 h of temperature acclimationat 25°C in both experiments; Fig. 1; Supplemental Fig.S1), the cells started proliferating with a growth ratecomparable to the control and reached a 2- to 3-fold

Figure 5. Low-temperature (77 K) fluorescence emission spectroscopy of the SDG fractions from the 25°C (C and D) and 42°C (Aand B) samples. The same volume (20 mL) of each fraction was mixed in 250 mL 60% glycerol, 10 mM HEPES-NaOH, pH 7.5before freezing in liquid nitrogen. The samples were excited at 435 nm for chlorophyll a (A and C) or 600 nm for phycocyanin (Band D), and the fluorescence emission was recorded from 600 to 800 nm or 620 to 800 nm, respectively. All spectra werenormalized to the spectrum baseline at 600 or 620 nm.










higher cell density but showed significantly less drymass per cell in late stationary phase (data not shown),indicating that the phycobilisomes in C. merolae mayfunction as a nutrient reserve, allowing for recycling ofamino acids into proteins needed in the acclimationprocess as already described for cyanobacteria understress conditions and nitrogen deprivation (Grossmanet al., 1993; Allen, 1984, Richaud et al., 2001; Görl et al.,

1998). The growth to a higher than normal cell density isa clear advantage in an environment where over-growing competitors is essential for survival.

An important factor to be considered when investi-gating the thermal acclimation is the temperature co-efficient (Q10) for biochemical reactions. Although theQ10 is not constant, for most temperature-dependentreactions it is assumed to be 2, which means that the

Figure 6. Mass spectrometrically determined protein intensities throughout the SDGs of the 15N-labeled 25°C and the 14N-la-beled 42°C sample. Same volume of the corresponding 14N and 15N fractions was tryptically digested in the same filter deviceaccording to the FASP method, and the resulting peptides were analyzed by LC-MS/MS. Peptide identification was performed byOMSSA and X! Tandem and protein intensities were quantified by qTrace.


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reaction rate will increase 2-fold for each 10°C increasein temperature (Atkin and Tjoelker, 2003). Thus, bio-chemical reactions in the 25°C C. merolae cultureshould run slower, unless the amount of enzymes in-volved in crucial temperature-dependent processes isincreased.On the other hand, all quantified photosystem pro-

teins (except the unchanged amount of the ATP syn-thase units) were found to be diminished in the25°C conditions (Fig. 2D), but the chlorophyll-to-photosystem ratio remained unchanged, since whenloading the same chlorophyll amount, the quantity ofPSI and PSII in both conditions is comparable (Fig. 6;Supplemental Fig. S3). All identified antenna proteinswere among the top 20 down-regulated proteins (Fig. 2,C and G). Furthermore, the different levels of down-regulation (the distal to the core phycocyanin wasmuch stronger affected than the core-forming allophy-cocyanin, which is in accordance with the differentialregulation of the linker proteins) indicated an alterationin the PBS composition, which is described as chromaticadaptation andnormally occurs as a response to changinglight qualities (Grossmann et al., 1993; Allen 1984), butalso to other environmental changes.Interestingly, the quantitative proteomics results in-

dicated that the PSI-specific antenna is more stable andless affected by the lower temperature. In line with thisobservation, the linker proteins were also differentlyaffected. The most severely down-regulated linker wasthe CMP166C phycocyanin-associated rod linker pro-tein, while the CpcG phycobilisome rod-core linkerpolypeptide (CMV051C) was less affected (Fig. 2C).These results are confirmed also on isolated thylakoids(Fig. 6; Supplemental Fig. S3). The CpcG linker is dis-cussed to be a part of a PSI-specific antenna, whichcontains further only phycocyanin but no allophyco-cyanin (Busch et al., 2010). In Synechocystis sp. PCC6803 CpcG2 rather than CpcG1 links phycocyanin rodsto PSI to form the PSI-specific antenna (Kondo et al.,2007). In the red alga genome, only one CpcG-likesubunit is present (Matsuzaki et al., 2004). Althoughthis subunit is missing the hydrophobic motif, which istypical for the CpcG2 of Synechocystis sp. PCC 6803, it isenriched within the PSI-linked phycobilisome sub-complex and is able to transfer energy to PSI (Buschet al., 2010). The separation of the thylakoidmembraneson the Suc gradient suggested that this linker poly-peptide comigrates together with PSI and LHC (Fig. 6;Supplemental Fig. S3). A further peak of the CpcGlinker was observed in the PBS fractions (10–14; Fig. 6;Supplemental Fig. S3), where it was much strongerdown-regulated at the lower temperature compared tothe amount in the PSI fractions. This again is anotherhint that the remodeling process did not affect thePSI-specific antenna as much as the PSII-associatedphycobilisome proteins. It is further confirmed by theobservation that in the PSI-containing fractions theamount of phyco- and allophycocyanin was onlyslightly reduced at 25°C compared to the PBS and PSIIfractions.

In contrast to photosynthesis-related proteins, mostof the identified proteins involved in cellular respira-tion were either expressed at similar levels or slightlydown-regulated at the lower temperature (Fig. 2E).Besides ATP generation, respiration within the mito-chondrion plays an important role in the maintenanceof the redox state within the chloroplast (for review, seeHoefnagel et al., 1998), which always tends to getoverreduced, as the rate of photochemical reaction andutilization of reducing potential in metabolism havebeen estimated to differ by at least 15 orders of mag-nitude (Huner et al., 1998). Since the enzymatic reac-tions at 25°C are slower, the electron sinks are limited,which leads to overreduction of the chloroplast. Theexport of malate from the chloroplast plays a significantrole in the transfer of reducing equivalents formed inexcess (Padmasree et al., 2002). Accordingly, additionalsink of reducing equivalents could be provided by theup-regulated cytosolic malate dehydrogenase (Fig. 2E).In line, the amount of ferredoxin-NADP+ reductase(FNR) was unchanged; while subunits of the photo-systems were strongly decreased, polypeptides of thecytochrome b6f complex were only slightly diminished(Fig. 2; Supplemental Table S1). The only slightly re-duced amount of ATP synthase, as well as the higherPSI-to-PSII ratio (Fig. 2D) may point to higher cyclicelectron flow around PSI.

The abundance of up-regulated stress-related proteinsindicates that a decrease of temperature is a stress factorfor the thermophilic alga. Protein folding and proteinhomeostasis were impaired at 25°C. This is not surpris-ing, as also for bacteria it was shown that chaperones(hence protein folding) were the rate-limiting factor forgrowth at lower temperatures (Ferrer et al., 2003).

Furthermore, there are many proteins increased inabundance at 25°C involved in the regulation of geneexpression on transcriptional and translational level.This is reasonable, since the colder temperature slowsdown the reaction rate (Q10 effect), which can be com-pensated by a higher amount of the correspondingproteins. Increased amounts of proteins involved inamino acid synthesis (vitamin B12 independent Metsynthase, Asp kinase) and chlorophyll biosynthesis(Mg protoporphyrin IX chelatase) together with resultson cell growth and photosynthesis at 25°C (Figs. 1, 3,and 5) point to active protein and chlorophyll biosyn-thesis under the lower temperatures. Here it is of notethat themaximumphotosynthetic efficiency of the 25°Cculture required a 2.5 times higher light intensity toachieve maximum photosynthetic efficiency as theculture grown at 42°C (Fig. 3) due to the reduced light-harvesting antenna size. On the other hand, with re-spect to chlorophyll, the oxygen evolution rate in the25°C culture was 2-fold higher than in the 42°C culture.

Interestingly, the photosynthetic apparatus ofC. merolae exhibits a remarkable stability over a greattemperature spectrum.Not only it is able to acclimate tothe colder temperature by decreasing the size and al-tering the composition of the PBS antenna, but once therearrangement is completed and the 25°C cells are again










shifted to 42°C, the photosynthesis is active andoxygen isevolved (Fig. 3). From biotechnological perspective, thegreat thermostability of the photosystems provides aninteresting system for heterologous expression of bio-technological products, which takes advantage of theaccelerated reaction rate at higher temperatures (Q10 ef-fect). The loss of the PBS antenna allows furthermore theattachment of recombinant proteins to the thylakoidmembranes. The majority of enzymes are active at tem-peratures higher than 25°C. As a soil-dwelling organismthe green alga Chlamydomonas reinhardtii, for example, israrely exposed to high temperatures. The temperatureoptimum of its hydrogenase, however, is at 60°C (Happeand Naber, 1993).

In conclusion, our data reveal that C. merolae accli-mates to suboptimal growth temperature by remodelingits photosynthetic apparatus, by tuning its photosyn-thetic capacity, and by adjusting its machinery for pro-tein folding, degradation, and homeostasis.

MATERIALS AND METHODS

Cultivation of Cyanidioschyzon merolae

The red algae Cyanidioschyzon merolae 10D was kindly provided by ProfessorTsuneyoshi Kuroiwa (Department of Life Science, Rikkyo University, Tokyo,Japan). Itwas cultivated in 23Allen’smediumas described byMinoda et al. (2004)at 42°C with 100 rpm shaking under continuous light of 70 mmol photons m22 s21.For studies on the influence of the temperature on the alga, one liter of themediumwas inoculated with liquid 42°C culture and split into two 2-L cotton-pluggedflasks and incubated at 25°C or 42°C. Culture inoculated with 25°C cells weretermed long-term adapted 25°C culture. Every 72 h 1mL of each culturewas takenand employed for flow cytometry measurements. For all of the performed exper-iments, exponentially growing cells were employed. For isotopic 15N labeling, theexponentially growing 42°C cells had to be diluted in 15N medium at least once.

All other experiments were performed on cultures in the exponential growthphase (approximately 1 month after inoculation).

Flow Cytometry

Growth of theC.merolae cultures at 25°C and 42°Cwasmonitored bymeans ofa flow cytometer (BD FACS Canto II) on two biological replicates in two inde-pendent experiments (Fig. 1; Supplemental Fig. S1). Laser voltage was set asfollows: forward-scatter (FSC), 645; side-scatter, 480; fluorescein isothiocyanate,280; Pacific blue, 620; Allophycocyanin, 290; PerCP-Cy5-5, 374. Fifty thousandevents (cells and bead reference) were recorded at a low to medium flow rate,identified by the FSC and side-scatter characteristics values, acquired in linearmode. For discrimination of living from dead cells, the fluorescent dye DAPI wasemployed, whose fluorescence was detected by the Pacific blue signal. Five mi-croliters of the culture were mixed with 23 Allen’s medium and DAPI at a finalconcentration of 2mg L21 andAlexa Fluor 488-labeled standard particles solution(Polyscience, 4 mm microspheres), adjusted to a concentration of 1.2*105 mL21

(30,000 standard particles in the mixture). Chlorophyll fluorescence was followedusing the PerCP-Cy5-5 filter settings (excitation at 488 nm, detection at 695 nm).Phycocyaninfluorescencewasmeasuredusing theAllophycocyaninfilter settings(excitation at 633 nm, detection at 660 nm). Fluorescence intensities were acquiredat log scale. All flow cytometry data were analyzed with the software BDFACSDiva, Version v6.12. Cellular debris with low FSC characteristics and deadcells (DAPI-positive) were excluded from further evaluation. Absolute numbersof cells in individual samples were calculated according to N (vital cells) = events(vital cells) * number (standard beads)/events (standard beads).

Oxygen Evolution Measurements

Oxygen evolution and consumption measurements were performed using aClark-type oxygen electrode as described in Naumann et al. (2007). The

electrode was calibrated using dithionite. Samples were dark incubated for20 min either at 25°C or 42°C before each measurement, which was carriedout at 42°C in the dark or at different light intensities (from 50 to 500 mmolphotons m22 s21).

Protein Separation and Immunoblot Analysis

Whole-cell samples (50mg total protein,measured by Pierce BCAProteinAssay Kit) were analyzed by discontinuous 13% (w/v) sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according toLaemmli (1970) and either stained with Coomassie Brilliant Blue or trans-ferred to nitrocellulose membrane (Hybond ECL membrane, Amersham),which was incubated with antibodies against the ATPase subunit b (ATPB)(1: 10,000 dilution, obtained from Agrisera) and against the phycocyaninalpha (CPCA) and beta (CPCB) chain (1: 5,000 dilution). After followingincubation with an anti-rabbit peroxidase-conjugated antibody, the signalswere detected by enhanced chemical luminescence. After staining, singleSDS-PAGE bands were excised and tryptic in-gel digested, prior to massspectrometric analysis.

Isolation of Thylakoid Membranes

Thylakoidmembranes of 25°C and 42°C C. merolae cultures (14N aswell as 15Nlabeled) were isolated as described previously (Chua and Bennoun, 1975;Petroutsos et al., 2009), breaking the cells by passing them two times through aself-made bio-nebulizer at a pressure of 20 psi. The thylakoids were eithertreated with NaBr (+NaBr samples) or not (2NaBr samples) and solubilizedwith n-dodecyl b-D maltoside prior to an ultracentrifugation on a linear Sucgradient (as described in Hippler et al. (1997) and in Busch et al. (2010)).

NaBr Treatment of Isolated Thylakoids

Thylakoids were resuspended in buffer (300 mM Suc, 50 mM Tris/HCl, pH7.5, 5 mM MgCl2, 10 mM NaCl) to a concentration of 2 mg ml21 chlorophyll, andmixed with NaBr (2 M final concentration). After incubation on ice for 30 min,themixturewas diluted 1:2with the same buffer and centrifuged at 10,000g, 4°Cfor 10min. The resulting pellet was resuspended in 5mMHEPES, pH 7.5, 10mM

EDTA and washed twice.

SDG Ultracentrifugation

Solubilized thylakoid membranes (0.8 mg ml21 chlorophyll) from 25°C and42°C cultures were loaded on separate discontinuous Suc density gradientswith Suc concentrations from 1.3 to 0.1 M (0.05% b-DM) and ultracentrifuged at33,000 rpm for 14 to 16 h using an SW41Ti rotor (Beckmann) at 4°C (Takahashiet al., 2006). SDGs were fractionated from bottom to top (approximately 300 mLper fraction).

Low-Temperature (77 K) FluorescenceEmission Spectroscopy

Twenty microliters from each SDG fraction were mixed with 60% glycerol,10 mM HEPES, pH 7.5, and frozen in liquid nitrogen. Low-temperature fluo-rescence emission spectra were recorded with the FP-6500 spectrofluorometer(Jasco). The samples were excited at 435 nm for chlorophyll a or 600 nm forphycocyanin, and thefluorescence emissionwas recorded from 600 to 800 nmor620 to 800 nm, respectively. All spectra were normalized to the PSI signal at728 nm.

FASP of SDG Fractions

Protein concentration of each SDG fraction was determined using the PierceBCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’sinstructions. For label-free quantification, the same volume of each fraction wasloaded on separate Amicon Ultra 0.5 mL centrifugal filters (30-kD cutoff; Mil-lipore) according to the FASPmethod (Wi�sniewski et al., 2009, 2011) withminormodifications. Same volume of each 14N and 15N fraction was trypticallydigested in one filter device. Proteins were reduced with 100 mM dithiothreitolin 100 mM Tris-HCl (pH 8.5) and 8 M urea (UA) at room temperature for 30 min.Subsequently, excess dithiothreitol was removed by buffer exchange using UA.


Nikolova et al.




Alkylation of Cys residues was performed by adding 50 mM iodoacetamide inUA followed by incubation for 20 min in the dark. Afterward, samples werewashed each three times with UA and 50 mM NH4HCO3. Following trypticaldigestion overnight (enzyme-to-protein ratio 1:50), peptides were eluted fromthe filter by centrifugation, acidified with 20 mL of 2% (v/v) formic acid, anddried by vacuum centrifugation.

LC-MS/MS and Data Analysis

Whole Cells

Whole-cell extracts of exponentially growingC.merolae cultures (25°C versus42°C) were mixed on equal protein amount [50 mg from each condition; the42°C culture was isotopically labeled with heavy nitrogen (15N)] and separatedby SDS-PAGE. Each gel lane was cut into up to 50 bands. Gel slices werein-gel-digested using trypsin, and resulting peptides were subjected to liquidchromatography coupled with high-resolution mass spectrometry. Only pro-teins identified and quantified in all three sets were further analyzed. Threedifferent protein sets were analyzed: protein extracts from 42°C culture mixedwith 25°C culture switched from 42°C (set1), a second biological replicate of thesame conditions (set3), and a mix of 42°C culture with long-term adapted 25°Cculture (set2). MS analysis, peptide identification (using Uniprot referenceproteome database of C. merolae, ProteomeId UP000007014), determination offalse discovery rates, and protein quantification were performed as describedby Wiemann et al. (2013).

SDG Fractions

Protein identification and relative quantification were performed label-free on SDG fractions from 25°C versus 42°C cultures (first biological rep-licate), as well as on 14N-labeled 42°C versus 15N-labeled 25°C fractions(second biological replicate). Additionally, the +NaBr as well as the2NaBrsamples from both biological replicates were mass spectrometricallyanalyzed.

Liquid chromatography was performed on an Ultimate 3000 nanoRSLCsystem (Thermo Scientfic) coupled via a nanospray interface to aQ ExactivePlus mass spectrometer (Thermo Scientific). An estimated 2 mg of peptideswere loaded on a trap column (C18 PepMap 100, 300 mM 3 5 mm, 5 mmparticle size, 100 Å pore size; Thermo Scientific) using 0.05% (v/v) tri-fluoroacetic acid/2% (v/v) acetonitrile and desalted for 2 min at a flowrate of 40 mL/min. For peptide separation, an Acclaim Pepmap capillarycolumn (75 mm 3 15 cm, 2 mm particle size, 100 Å pore size; Thermo Sci-entific) was used. Mobile phases consisted of 0.1% (v/v) formic acid inultrapure water (A) and 0.1% (v/v) formic acid/80% (v/v) acetonitrile inultrapure water (B). Gradient elution was carried out as follows: 2.5% to30% B over 93 min, 30% to 50% over 7 min, 50% to 99% B over 3 min, 99% Bover 10 min.

The mass spectrometer was operated in a data-dependent mode that au-tomatically switched between one survey scan (m/z 375–1400, resolution70,000 atm/z 200, automatic gain control target value 1e6, maximum injectiontime 30 ms) and up to 12 higher-energy C-trap dissociation fragmentationscans on the 12 most intense ions (27% normalized collision energy, resolu-tion 17,500 at m/z 200, automatic gain control target value 1e5, underfill ratio1%, maximum injection time 64 ms, dynamic exclusion 20 s, precursor iso-lation window 1.5 m/z).

Label-Free QuantificationMSdatawere searched against theUniprot referenceproteome database of C. merolae (ProteomeId UP000007014) using MaxQuant(Version 1.5.3.30; Cox and Mann, 2008). Default search and quantification set-tings were applied with the following exceptions: “match between runs” wasactivated and protein decoy sequences were generated by randomization. Dueto the heterogenous nature of SDG fractions, nonnormalized protein intensitiesinstead of label-free quantification intensities were used for the preparation offigures.

Quantification of 14N/15N-Labeled Proteins MS raw files were converted tomzML format with msconvert (Proteowizard version 3.0.7692; Kessner et al.,2008). Peptide identification and quantification was carried out essentially asdescribed for labeled whole cells. Instead of computing 14N/15N intensity ra-tios, protein intensities for each labeling state were calculated by summing uppeptide intensities.

MS data have been deposited to ProteomeXchange via the PRIDE partnerrepository (Vizcaíno et al., 2016) with the dataset identifier PXD005615.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Growth and pigment fluorescence monitoring ofC. merolae 25°C and 42°C cultures (second biological replicate).

Supplemental Figure S2. Low-temperature (77 K) fluorescence emissionspectroscopy of the SDG fractions from the 25°C (E, F, G, H) and 42°C(A, B, C, D) samples.

Supplemental Figure S3. Mass spectrometrically determined protein in-tensities throughout the SDGs of the label-free 25°C and 42°C sample.

Supplemental Table S1. Additional ratios of proteins identified in two ofthe three quantified protein data sets.

Received January 30, 2017; accepted March 4, 2017; published March 7, 2017.

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