TY - JOUR
AU - Hippler, Michael
AB - Abstract The thermophilic alga C. merolae thrives in extreme environments (low pH and temperature between 40°C and 56°C). In this study, we investigated the acclimation process of the alga to a colder temperature (25°C). A long-term cell growth experiment revealed an extensive remodeling of the photosynthetic apparatus in the first 250 h of acclimation, which was followed by cell growth to an even higher density than the control (grown at 42°C) cell density. Once the cells were shifted to the lower temperature, the proteins of the light-harvesting antenna were greatly down-regulated and the phycobilisome composition was altered. The amount of PSI and PSII subunits was also decreased, but the chlorophyll to photosystems ratio remained unchanged. The 25°C cells possessed a less efficient photon-to-oxygen conversion rate and require a 2.5 times higher light intensity to reach maximum photosynthetic efficiency. With respect to chlorophyll, however, the photosynthetic oxygen evolution rate of the 25°C culture was 2 times higher than the control. Quantitative proteomics revealed that acclimation requires, 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 demands placed on the system and revealed the capability of C. merolae to grow under a broad range of temperatures. Extremophilic organisms like the members of the rhodophytan order Cyanidiales occupy harsh ecological niches, being able to withstand very low pH conditions (pH 0.5–3) and relatively high temperatures (40°C–56°C). Phylogenetic studies indicate that they are the most ancient clade of red algae having diverged at the 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 in these hostile environments are mostly prokaryotes. However, with varying ecological conditions, Cyanidiales are able to persist at temperatures as low as approximately 35°C in aquatic sites or 10°C in soil, because below these temperature limits, there is too much competition with other acidophilic algae (Doemel and Brock, 1971; Reeb and Bhattacharya, 2010). Cyanidioschyzon merolae, one of the two Cyanidiales with a fully sequenced genome (Ohta et al., 2003; Matsuzaki et al., 2004; Nozaki et al., 2007), is an important eukaryotic model organism because of its unique genomic and proteomic characteristics. With its 16.5 Mbp, the genome of C. merolae has one of the smallest sizes for a photosynthetic eukaryote. Furthermore, it has a high degree of gene compaction since it possesses only 0.5% intron-containing protein genes and low gene redundancy (Ohta et al., 2003; Matsuzaki et al., 2004). This and the very small proteome allow studies on the origin, evolution, primary, and secondary endosymbiosis as well as complex fundamental processes of eukaryotic cells (Matsuzaki et al., 2004). Furthermore, the C. merolae cell has the simplest structure among all photosynthetic eukaryotes with no cell wall and no vacuole, one nucleus, one mitochondrion, and one chloroplast as well as a reduced set of other ultrastructural components (Merola et al., 1981; Kuroiwa et al., 1994). C. merolae is intriguing also regarding the evolution of the photosynthetic machinery. In prokaryotic cyanobacteria, the antenna system consists of solely chlorophyll a and phycobilisomes (PBSs), which were lost multiple times in the course of eukaryotic evolution and replaced by thylakoid membrane-integral light-harvesting complexes (LHCs) in green algae and higher plants (Neilson and Durnford, 2010). Containing the unique combination of both antenna types, Rhodophyta represents an important evolutionary intermediate. Besides phycobilisomes the members of the group encode a species-dependent number of chlorophyll a-binding LHCs, which functionally associate solely with PSI (Wolfe et al., 1994; Busch et al., 2010). Phycobilisomes greatly expand the solar spectrum used for photosynthesis, because they absorb in the spectral gap between the blue and main-red absorption of chlorophyll. Located on the outer surface of the thylakoid membrane, the PBSs are assembled from various types of phycobiliproteins, which have covalently bound light-absorbing tetrapyrrole chromophores, and from nonpigmented linker polypeptides, which are involved in the assembly and stability of the complex at different levels, but also facilitate efficient flow of excitation energy. Four major subgroups of phycobiliproteins have been described: allophycocyanin, at the membrane surface, forms the core structure of the PBS, whereas the series of rods radiating out from the core is composed of phycocyanin (closest to the core), and, distal to the core, phycoerythrin and phycoerythrocyanin (latter one is absent in red algae; Adir, 2005). Furthermore, C. merolae and the closely related red alga Galdieria sulphuraria also lack phycoerithrin (responsible for red coloring); thus, the algae appear blue-green in color (Stadnichuk et al., 2011). Light energy is absorbed by the distal end of the rods and transferred toward allophycocyanin, which can supply excitation to the chlorophyll pigments in the photosynthetic reaction centers. The basic core-rod structure is widely conserved among cyanobacteria and algae, but there is a great diversity across different species regarding phycobiliproteins, core structure type, and linker polypeptides (rod linkers and rod-core linkers). The diversity of the latter is important for the structural diversity of peripheral rods (Kondo et al., 2007). Although PBSs are primarily attached to PSII, the presence of functional, specific phycobilisome PSI antenna in cyanobacteria and C. merolae was demonstrated. Kondo et al. (2007) identified two types of PBS in the cyanobacterium Synechocystis sp. PCC 6803, differing in their rod-core linkers (CpcG1- and CpcG2-PBS) and demonstrated a 3-fold higher efficiency from CpcG2-PBS to PSI than to PSII. Busch et al. (2010) also showed a PSI-specific phycobilisome subcomplex in C. merolae, which is able to transfer excitation energy to PSI. It lacks allophycocyanin and contains phycocyanin and a CpcG-like rod-core linker polypeptide. Recently, a megacomplex containing PBS, PSII, and PSI was isolated from the cyanobacterium Synechocystis PCC 6803 after in vivo protein cross linking, demonstrating that PBS can supply excitation energy to both photosystems (Liu et al., 2013). In the megacomplex, four PSII subunits (PsbB, PsbC, PsbD, PsbI) are linked to the phycobilisome linker polypeptide ApcE, whereas ApcD is located on the edge area of PSI through a cove formed by PsaD and PsaA (Liu et al., 2013). During dark-light transitions, however, such PBS-PSII-PSI megacomplexes are not active in vivo, but modulation of the excitation energy transfer to the photosystems is regulated by functional uncoupling of PBS from the PSI and almost no reattachment to PSII (Chukhutsina et al., 2015). Since the mobility of the most transmembrane proteins in the thylakoid membrane is very restricted, mobility of the extrinsic antenna is thought to play an important role in photoprotection in cyanobacteria and mesophilic red algae by the process of state transition (Kaňa et al., 2014; Kirilovsky, 2015). In thermophilic algae, however, excess light is dissipated via the process of nonphotochemical quenching as the main photoprotective mechanism (Krupnik et al., 2013). The extremophilic alga C. caldarium exhibits highly restricted phycobilisome mobility, which is due to the strength of the PBS-photosystem interaction rather than to macromolecular crowding and a decrease in lipid desaturation (Kaňa et al., 2014). The latter is considered to be key in acclimation process to lower temperatures in mesophilic and to some extent in thermophilic cyanobacteria (Kiseleva et al., 1999; Murata and Los, 1997). However, increased PBS mobility in the thermophilic alga Cyanidium caldarium could be achieved by lowering the growth temperatures, which leads to weaker PBS-photosystem binding (Kaňa et al., 2014). In this study, we investigated the acclimation of the thermophilic alga C. merolae to lower temperatures (25°C versus 42°C). Interestingly, our data revealed that C. merolae is well capable of growing at 25°C. Growth at this temperature, however, is accompanied by a substantial remodeling of the photosynthetic machinery. RESULTS Herein we investigated the ability of the thermophilic alga C. merolae to acclimate to colder temperatures. Under Temperature Stress, the Alga Started Growing Once the Remodeling of the Photosynthetic Machinery Was Completed and Outgrew the Control To monitor and compare the growth behavior of the alga, two cultures emanating from a 42°C control culture were grown at 25°C and 42°C, respectively. Cell density and the fluorescence of cyanobilin and chlorophyll were recorded via flow cytometry in two independent experiments (Fig. 1; Supplemental Fig. S1). For further evaluation, only the data of living cells (4,6-diamidino-2-phenylindole [DAPI] negative) were considered. Shifting of cells to a colder temperature resulted in an immediate and steady decrease of the cyanobilin and chlorophyll fluorescence (Fig. 1; Supplemental Fig. S1). However, low fluorescence intensities remained almost unchanged in the course of the experiment. Cells cultivated at 42°C on the contrary showed an overall continuous increase in pigment fluorescence. Figure 1. Open in new tabDownload slide Growth and pigment fluorescence monitoring of C. merolae 25°C and 42°C cultures (first biological replicate). Shown is the average of 1 mL triplicates of each culture, which were measured every 72 h via flow cytometry. DAPI was employed for discrimination of dead cells. Cell density of DAPI-negative cells (designated to be alive) and the mean pigment fluorescence signal per cell are shown. Chlorophyll was excited 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 was detected at 660 nm. Figure 1. Open in new tabDownload slide Growth and pigment fluorescence monitoring of C. merolae 25°C and 42°C cultures (first biological replicate). Shown is the average of 1 mL triplicates of each culture, which were measured every 72 h via flow cytometry. DAPI was employed for discrimination of dead cells. Cell density of DAPI-negative cells (designated to be alive) and the mean pigment fluorescence signal per cell are shown. Chlorophyll was excited 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 was detected at 660 nm. After an approximately 250 h of temperature acclimation, the 25°C cells started growing with a growth rate comparable to the control and reached a 2- to 3-fold higher cell density (Fig. 1; Supplemental Fig. S1). Quantitative proteomics on whole cells revealed down-regulation of antenna proteins, PSI, and PSII subunits. For detailed studies of the acclimation process, quantitative proteomics was employed. Cells grown at 42°C were metabolically labeled with heavy nitrogen, whereas the 25°C cells were grown in normal 14N-labeled medium. Three different protein sets were analyzed: a mix of 42°C and 25°C protein extract (two independent biological replicates, designated set1 and set3) and a protein mix of 42°C and 25°C long-term adapted culture (designated set2; see “Materials and Methods”). As indicated by the pigment fluorescence in the growth monitoring experiment, all identified light-harvesting antenna proteins were down-regulated at 25°C compared to the control (Fig. 2C; Supplemental Table S1). The PBS antenna proteins (phycocyanin, allophycocyanin, and the linker polypeptides) were much more affected (down to 2%–10%) than the chlorophyll containing LHC proteins (CMN235C, CMQ142C, down to 10%–20%). Furthermore, phycocyanin-binding proteins were stronger down-regulated than allophycocyanin-binding proteins, as was the phycocyanin-associated rod linker protein (CMP166C) compared to the phycobilisome linker (CMV157C, ApcE) and rod-core linker (CMV051C, CpcG) polypeptides (Fig. 2C). The fact that the different chains of phyco- and allophycocyanin-binding proteins were quantified with similar ratios among each other strongly supports the data. All of these identified phycobilisome antenna proteins were among the top 20 down-regulated proteins (Fig. 2G). Figure 2. Open in new tabDownload slide Immunoblot and comparative quantitative proteomic analysis of C. merolae whole cells grown at 25°C or 42°C. A, Immunoblot analysis of 30 μg total protein extract samples reveals the severe down-regulation of phycocyanin α- and β-chain (antiphycocyanin antibody) in the culture grown at suboptimal temperature. ATPase subunit b (ATPB) served as loading control. B, Key to heat map. The heat map represents the log2 ratios of the protein ranges, from black to green indicating an up-regulation, and from black 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 on equal protein amount (50 μg) and separated by SDS-PAGE. The resulting bands were analyzed by LC-MS/MS. Three different protein sets were analyzed: 42°C culture mixed with 25°C culture switched from 42°C (1), a second biological replicate of the same 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 2. Open in new tabDownload slide Immunoblot and comparative quantitative proteomic analysis of C. merolae whole cells grown at 25°C or 42°C. A, Immunoblot analysis of 30 μg total protein extract samples reveals the severe down-regulation of phycocyanin α- and β-chain (antiphycocyanin antibody) in the culture grown at suboptimal temperature. ATPase subunit b (ATPB) served as loading control. B, Key to heat map. The heat map represents the log2 ratios of the protein ranges, from black to green indicating an up-regulation, and from black 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 on equal protein amount (50 μg) and separated by SDS-PAGE. The resulting bands were analyzed by LC-MS/MS. Three different protein sets were analyzed: 42°C culture mixed with 25°C culture switched from 42°C (1), a second biological replicate of the same 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. Additionally, most of the photosystem subunits were down-regulated at the lower temperature (Fig. 2D; Supplemental Table S1). The subunits of the ATP synthase, FNR as well as ferredoxin and components of the cytochrome b6f complex were less affected compared to the PSI and PSII units (Fig. 2D; Supplemental Table S1). Four of the PSII subunits (D1 and D2 core proteins as well as PsbC and PsbO) are among the top 20 down-regulated proteins (Fig. 2G). Furthermore, proteins involved in the stability and biogenesis of PSII were also slightly down-regulated (Fig. 2D; Supplemental Table S1). The majority of the proteins involved in different cellular respiration processes were similarly expressed in both conditions (Fig. 2E; Supplemental Table S1). The enzymes controlling the first phase of glycolysis were stronger affected by the lower temperature (2-fold down-regulation) than the ones of the last two steps of the pathway. The identified proteins of the Krebs cycle were differently regulated. The identified subunits of the mitochondrial ATP synthase showed no change in abundance at 25°C. Most of the identified stress-related proteins like proteins responsible for proper protein folding (chaperones and chaperonins, heat-shock proteins [proteins of both of the Hsp70 and Hsp90 families]), quality control (calnexin), and degradation of wrongly folded proteins (ubiquitin- carboxyl-terminal hydrolase, ubiquitinyl hydrolase 1, ubiquitin-activating enzyme) were up-regulated in cells grown at 25°C (Fig. 2F; Supplemental Table S1). HSP90, ubiquitin-activating enzyme and chloroplast chaperonin CPN60 were furthermore among the top 20 up-regulated proteins (Fig. 2H). Interestingly, whereas a protein similar to thioredoxin h was up-regulated (Supplemental Table S1), proteins directly involved in reactive oxygen species (ROS) defense (catalase and superoxide dismutase) were down at least by 50%. The most affected protein by the low temperature in all three sets was the phycocyanin-associated rod linker protein CMP166C (25°C/42°C ratio: 0.029 ± 0.01). Furthermore, besides all identified antenna proteins and four PSII and two PSI subunits, among the top 20 down-regulated proteins were also the steroid mono-oxygenase (CML339C), the Mn superoxide dismutase (CMT028C) and three hypothetical proteins (CMP346C, CMJ121C, and CMH239C; Fig. 2G). Although quantified in two of the three sets among the top 20 down-regulated proteins is also the CpcG PBS rod-core linker polypeptide with a 23-fold down-regulation (Supplemental Table S1). Among the most up-regulated proteins were, besides many stress-related proteins, also proteins involved in nucleic acid binding and the modification of nucleic acid structures (CMM134C uncharacterized protein, polyadenylate-binding protein, RNA helicase, DNA gyrase, and exodeoxyribonuclease; Fig. 2H; Supplemental Table S1). Furthermore, the translational elongation factor eIF-4G was found to be highly up-regulated, as were enzymes involved in the amino acid biosynthesis (aspartokinase, vitamin B12 independent Met synthase, adenosylhomocysteinase) and catabolism (2-amino-3-ketobutyrate coenzyme A ligase). Although quantified in one of the three sets highly up-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-Efficient Photon-to-Oxygen Conversion Rate Cells from both conditions were collected from the exponential growth phase and subjected to an oxygen electrode. Cells were dark incubated for 20 min either at 25°C or 42°C before each measurement, which was carried out at 42°C in the dark in order to measure O2 consumption or at light intensities from 50 to 500 µmol photons m−2 s−1 to record O2 evolution. When equalized on the same cell number, the 42°C culture exhibits two to three times higher oxygen evolution depending on the light intensity (Fig. 3B) and very similar respiration capacity. With respect to chlorophyll, however, the photosynthetic oxygen evolution rate of the 25°C culture was two times higher compared to the control (Fig. 3A), even though the amount of chlorophyll and chlorophyll containing LHC proteins was strongly diminished (Fig. 2C). Figure 3. Open in new tabDownload slide Oxygen evolution and consumption rates of C. merolae cells grown at 25°C and 42°C and measured at light intensities from 50 to 500 µmol photons m−2 s−1. Exponentially growing cells were dark incubated for 20 min at the corresponding temperature before each measurement, which was performed using a Clark-type oxygen electrode at 42°C at different light intensities. Values are means ± sd of three independent biological replicates. A, Oxygen evolution and consumption per µg chlorophyll. B, Oxygen evolution and consumption per 109 cells. Figure 3. Open in new tabDownload slide Oxygen evolution and consumption rates of C. merolae cells grown at 25°C and 42°C and measured at light intensities from 50 to 500 µmol photons m−2 s−1. Exponentially growing cells were dark incubated for 20 min at the corresponding temperature before each measurement, which was performed using a Clark-type oxygen electrode at 42°C at different light intensities. Values are means ± sd of three independent biological replicates. A, Oxygen evolution and consumption per µg chlorophyll. B, Oxygen evolution and consumption per 109 cells. Furthermore, the control culture reached its maximum photosynthetic efficiency at lower light intensities, since its O2 evolution was already saturated at around 100 µmol photons m−2 s−1. The 25°C culture however showed a steady rise of the produced oxygen with increasing light intensities, which flattens out at 250 µmol photons m−2 s−1, corresponding to a 2.5 times higher light intensity that is needed to achieve maximum photosynthetic efficiency. Notably, this finding was similar to one observed in Synechocystis sp PCC 6803 strain lacking a PBS linker polypeptide (Shen et al., 1993). Spectroscopic and Mass Spectrometric Analysis on Isolated Thylakoids To investigate the acclimation of the photosynthetic apparatus of C. merolae, isolated thylakoid membranes were solubilized with detergent and fractionated via Suc density centrifugation (Fig. 4). Each fraction was analyzed by fluorescence spectroscopy (Fig. 5) and additionally digested with trypsin according to the filter-aided sample preparation (FASP) protocol (Wiśniewski et al., 2009). Peptides were then examined by liquid chromatography-tandem mass spectrometry (LC-MS/MS; Fig. 6). Figure 4. Open in new tabDownload slide SDGs of thylakoid membranes from C. merolae 25°C and 42°C cultures. Two cultures, emanating from the same 42°C culture, were grown at either 25°C or 42°C. Isolated thylakoid membranes of both samples, equalized on the same chlorophyll level, were solubilized with 0.9% β-DM and loaded on separate discontinuous SDGs with Suc concentrations from 1.3 to 0.1 m, and after ultracentrifugation they were fractionated from the bottom to the top. Figure 4. Open in new tabDownload slide SDGs of thylakoid membranes from C. merolae 25°C and 42°C cultures. Two cultures, emanating from the same 42°C culture, were grown at either 25°C or 42°C. Isolated thylakoid membranes of both samples, equalized on the same chlorophyll level, were solubilized with 0.9% β-DM and loaded on separate discontinuous SDGs with Suc concentrations from 1.3 to 0.1 m, and after ultracentrifugation they were fractionated from the bottom to the top. Figure 5. Open in new tabDownload slide Low-temperature (77 K) fluorescence emission spectroscopy of the SDG fractions from the 25°C (C and D) and 42°C (A and B) samples. The same volume (20 µL) of each fraction was mixed in 250 µL 60% glycerol, 10 mm HEPES-NaOH, pH 7.5 before freezing in liquid nitrogen. The samples were excited at 435 nm for chlorophyll a (A and C) or 600 nm for phycocyanin (B and D), and the fluorescence emission was recorded from 600 to 800 nm or 620 to 800 nm, respectively. All spectra were normalized to the spectrum baseline at 600 or 620 nm. Figure 5. Open in new tabDownload slide Low-temperature (77 K) fluorescence emission spectroscopy of the SDG fractions from the 25°C (C and D) and 42°C (A and B) samples. The same volume (20 µL) of each fraction was mixed in 250 µL 60% glycerol, 10 mm HEPES-NaOH, pH 7.5 before freezing in liquid nitrogen. The samples were excited at 435 nm for chlorophyll a (A and C) or 600 nm for phycocyanin (B and D), and the fluorescence emission was recorded from 600 to 800 nm or 620 to 800 nm, respectively. All spectra were normalized to the spectrum baseline at 600 or 620 nm. Figure 6. Open in new tabDownload slide Mass spectrometrically determined protein intensities throughout the SDGs of the 15N-labeled 25°C and the 14N-labeled 42°C sample. Same volume of the corresponding 14N and 15N fractions was tryptically digested in the same filter device according to the FASP method, and the resulting peptides were analyzed by LC-MS/MS. Peptide identification was performed by OMSSA and X! Tandem and protein intensities were quantified by qTrace. Figure 6. Open in new tabDownload slide Mass spectrometrically determined protein intensities throughout the SDGs of the 15N-labeled 25°C and the 14N-labeled 42°C sample. Same volume of the corresponding 14N and 15N fractions was tryptically digested in the same filter device according to the FASP method, and the resulting peptides were analyzed by LC-MS/MS. Peptide identification was performed by OMSSA and X! Tandem and protein intensities were quantified by qTrace. Changes of the pigment composition caused by the lower temperature were directly visible on the Suc gradients (Fig. 4). The intact phycobilisomes as large antenna complexes migrated to the high-density region of the gradient and appeared blue due to the phycobilins. Visual comparison of both gradients indicated that the PBS fraction was strongly diminished in 25°C cells. Furthermore, the fractions containing PSI, PSII, and PBS were excited at 435 nm for chlorophyll a (Fig. 5, A and C) or at 600 nm for phycocyanin-dependent excitation energy transfer (Fig. 5, B and D). The fluorescence emission was recorded from 600 to 800 nm or 620 to 800 nm, respectively, and all spectra were normalized to 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 energy transfer within intact PBS was indicated by the low phyco- (642 nm) and allophycocyanin (655 nm) fluorescence emission and the strong 681nm peak, originating from terminal energy acceptor ApcE (Busch et al., 2010; Su et al., 1992; Shen et al., 1993), which stabilizes the phycobilisome core architecture. In the 25°C culture the ApcE fluorescence emission was greatly reduced (Fig. 5D). The presence of a PSI emission peak, after excitation of chlorophyll a molecules, indicated remaining PSI in the phycobilisome fraction (Fig. 5C). With decreasing Suc density within the gradient, the emission spectra of the fractions revealed a dominating emission peak at 728 nm (fractions 18–21) when excited at 435 nm (Fig. 5, A and C), reflecting the fluorescence of red chlorophylls from PSI (Bruce et al., 1985; Busch et al., 2010; Kaňa et al., 2014). On the other hand, in fractions 21 to 23 the strong emission peaks at 686 nm and 692 nm suggested the presence of PSII. Thus, fractions 21 to 23 (and to some extent fraction 20 of the 25°C sample) contained both PSI and PSII. When excited at 600 nm (Fig. 5, B and D) the PSI enriched fractions (fractions 18–21) exhibited two phycobilin peaks at 642 nm and at 655 nm as well as the PSI peak at 728 nm, indicating that antenna proteins migrated with PSI at the same Suc density. This comigration was strongly diminished, when thylakoids were treated with NaBr prior to separation on the Suc gradients (Supplemental Fig. S2, D and H). For the 42°C as well as the 25°C sample, this led to a 2-fold reduction of the 642 nm and 655 nm peaks in the PSI fractions (Supplemental Figure S2, B, D, F, and H), whereas the fluorescence of the PSII antenna decreased around four times in the 42°C and 2 times in the 25°C culture. MS/MS analysis (Fig. 6) of the Suc fractions confirmed the spectroscopic data about the distribution of the protein complexes within the gradient and allowed the comparison of both cultures. In contrast to the mass spectrometric analysis of whole cells, where equal protein amount of both samples was employed, for this experiment, the samples were equalized to the same chlorophyll concentration prior to the sucrose density gradient (SDG) ultracentrifugation. For all further experiments (Figs. 5 and 6; Supplemental Figs S2 and S3), same volume hence same chlorophyll amount of the corresponding SDG fractions was employed. Thus, although the 25°C whole cell exhibited reduced PSI, PSII, and chlorophyll content (Figs. 1 and 2D) as well as light-harvesting proteins (Fig. 2C), when loading the same Chl amount on the gradient, PSI and PSII concentration was comparable in both conditions (Fig. 6). Furthermore, both photosystems peaked in different fractions: PSI was concentrated in fractions 18 to 22, whereas PSII in fractions 20 to 24 (Fig. 6). Light-harvesting proteins peaked similarly to PSI, which is even clearer in the second biological replicate (Supplemental Fig. S3). With respect to phyco- and allophycocyanin, a 2- to 4-fold down-regulation was present in the PBS fractions (fractions 10 to 15 of the SDGs) of the 25°C sample compared to the control, which was consistent with the whole-cell results. The sum of the protein intensity of all allophycocyanin subunits that migrated together with PSI and PSII, however, did not exhibit any significant change at the lower temperature (Fig. 6; Supplemental Fig. S3). Phycocyanin appeared to be more strongly impacted by the low temperature, which is more pronounced in the first biological replicate (Fig. 6) than the second (Supplemental Fig. S3). Furthermore, the impact was greater in the PSII fractions 22 and 23, as well as in fractions 24 to 26, where probably free proteins migrated, including free phycocyanin, since these fractions exhibited strong peak at 642 nm, when phycocyanin was excited (data not shown). Strong reduction of the linkers was observed in every biological and technical replicate in the 25°C sample (Fig. 6; Supplemental Fig. S3). Very strongly down-regulated was the phycocyanin-associated rod linker protein CMP166C, which in the control sample peaked in the low-density fractions 20 to 24 (Fig. 6; Supplemental Fig. S3). Less affected was the phycobilisome rod-core linker polypeptide CpcG, which has been discussed to be a part of the PSI-specific antenna (Busch et al., 2010). Consistently, CpcG rather comigrated with the PSI complex and with the free phycobiliprotein complex, where it was more diminished at 25°C as compared to its abundance in the PSI-related fractions. DISCUSSION The thermophilic red alga C. merolae has the capacity and flexibility to acclimate to a temperature of 25°C. This acclimation requires a pronounced remodeling of the photosynthetic machinery permitting growth to even higher cell densities. A first step toward this remodeling is a pronounced degradation of the phycobilisomes (Fig. 1; Supplemental Fig. S1) as revealed by the immediate and steady decrease of the phycobilisomes-associated pigment fluorescence and further confirmed by immunoblot analysis and mass spectrometry of whole cells (Fig. 2, A–H). Once the photosynthetic machinery was remodeled (after approximately 250 h of temperature acclimation at 25°C in both experiments; Fig. 1; Supplemental Fig. S1), the cells started proliferating with a growth rate comparable to the control and reached a 2- to 3-fold higher cell density but showed significantly less dry mass per cell in late stationary phase (data not shown), indicating that the phycobilisomes in C. merolae may function as a nutrient reserve, allowing for recycling of amino acids into proteins needed in the acclimation process as already described for cyanobacteria under stress conditions and nitrogen deprivation (Grossman et al., 1993; Allen, 1984, Richaud et al., 2001; Görl et al., 1998). The growth to a higher than normal cell density is a clear advantage in an environment where overgrowing competitors is essential for survival. An important factor to be considered when investigating the thermal acclimation is the temperature coefficient (Q10) for biochemical reactions. Although the Q10 is not constant, for most temperature-dependent reactions it is assumed to be 2, which means that the reaction rate will increase 2-fold for each 10°C increase in temperature (Atkin and Tjoelker, 2003). Thus, biochemical reactions in the 25°C C. merolae culture should run slower, unless the amount of enzymes involved in crucial temperature-dependent processes is increased. On the other hand, all quantified photosystem proteins (except the unchanged amount of the ATP synthase units) were found to be diminished in the 25°C conditions (Fig. 2D), but the chlorophyll-to-photosystem ratio remained unchanged, since when loading the same chlorophyll amount, the quantity of PSI and PSII in both conditions is comparable (Fig. 6; Supplemental Fig. S3). All identified antenna proteins were 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 was much stronger affected than the core-forming allophycocyanin, which is in accordance with the differential regulation of the linker proteins) indicated an alteration in the PBS composition, which is described as chromatic adaptation and normally occurs as a response to changing light qualities (Grossmann et al., 1993; Allen 1984), but also to other environmental changes. Interestingly, the quantitative proteomics results indicated that the PSI-specific antenna is more stable and less affected by the lower temperature. In line with this observation, the linker proteins were also differently affected. The most severely down-regulated linker was the CMP166C phycocyanin-associated rod linker protein, while the CpcG phycobilisome rod-core linker polypeptide (CMV051C) was less affected (Fig. 2C). These results are confirmed also on isolated thylakoids (Fig. 6; Supplemental Fig. S3). The CpcG linker is discussed to be a part of a PSI-specific antenna, which contains further only phycocyanin but no allophycocyanin (Busch et al., 2010). In Synechocystis sp. PCC 6803 CpcG2 rather than CpcG1 links phycocyanin rods to PSI to form the PSI-specific antenna (Kondo et al., 2007). In the red alga genome, only one CpcG-like subunit is present (Matsuzaki et al., 2004). Although this subunit is missing the hydrophobic motif, which is typical for the CpcG2 of Synechocystis sp. PCC 6803, it is enriched within the PSI-linked phycobilisome subcomplex and is able to transfer energy to PSI (Busch et al., 2010). The separation of the thylakoid membranes on the Suc gradient suggested that this linker polypeptide comigrates together with PSI and LHC (Fig. 6; Supplemental Fig. S3). A further peak of the CpcG linker was observed in the PBS fractions (10–14; Fig. 6; Supplemental Fig. S3), where it was much stronger down-regulated at the lower temperature compared to the amount in the PSI fractions. This again is another hint that the remodeling process did not affect the PSI-specific antenna as much as the PSII-associated phycobilisome proteins. It is further confirmed by the observation that in the PSI-containing fractions the amount of phyco- and allophycocyanin was only slightly reduced at 25°C compared to the PBS and PSII fractions. In contrast to photosynthesis-related proteins, most of the identified proteins involved in cellular respiration were either expressed at similar levels or slightly down-regulated at the lower temperature (Fig. 2E). Besides ATP generation, respiration within the mitochondrion plays an important role in the maintenance of the redox state within the chloroplast (for review, see Hoefnagel et al., 1998), which always tends to get overreduced, as the rate of photochemical reaction and utilization of reducing potential in metabolism have been estimated to differ by at least 15 orders of magnitude (Huner et al., 1998). Since the enzymatic reactions at 25°C are slower, the electron sinks are limited, which leads to overreduction of the chloroplast. The export of malate from the chloroplast plays a significant role in the transfer of reducing equivalents formed in excess (Padmasree et al., 2002). Accordingly, additional sink of reducing equivalents could be provided by the up-regulated cytosolic malate dehydrogenase (Fig. 2E). In line, the amount of ferredoxin-NADP+ reductase (FNR) was unchanged; while subunits of the photosystems were strongly decreased, polypeptides of the cytochrome b6f complex were only slightly diminished (Fig. 2; Supplemental Table S1). The only slightly reduced amount of ATP synthase, as well as the higher PSI-to-PSII ratio (Fig. 2D) may point to higher cyclic electron flow around PSI. The abundance of up-regulated stress-related proteins indicates that a decrease of temperature is a stress factor for the thermophilic alga. Protein folding and protein homeostasis were impaired at 25°C. This is not surprising, as also for bacteria it was shown that chaperones (hence protein folding) were the rate-limiting factor for growth at lower temperatures (Ferrer et al., 2003). Furthermore, there are many proteins increased in abundance at 25°C involved in the regulation of gene expression on transcriptional and translational level. This is reasonable, since the colder temperature slows down the reaction rate (Q10 effect), which can be compensated by a higher amount of the corresponding proteins. Increased amounts of proteins involved in amino acid synthesis (vitamin B12 independent Met synthase, Asp kinase) and chlorophyll biosynthesis (Mg protoporphyrin IX chelatase) together with results on cell growth and photosynthesis at 25°C (Figs. 1, 3, and 5) point to active protein and chlorophyll biosynthesis under the lower temperatures. Here it is of note that the maximum photosynthetic efficiency of the 25°C culture required a 2.5 times higher light intensity to achieve maximum photosynthetic efficiency as the culture grown at 42°C (Fig. 3) due to the reduced light-harvesting antenna size. On the other hand, with respect to chlorophyll, the oxygen evolution rate in the 25°C culture was 2-fold higher than in the 42°C culture. Interestingly, the photosynthetic apparatus of C. merolae exhibits a remarkable stability over a great temperature spectrum. Not only it is able to acclimate to the colder temperature by decreasing the size and altering the composition of the PBS antenna, but once the rearrangement is completed and the 25°C cells are again shifted to 42°C, the photosynthesis is active and oxygen is evolved (Fig. 3). From biotechnological perspective, the great thermostability of the photosystems provides an interesting system for heterologous expression of biotechnological products, which takes advantage of the accelerated reaction rate at higher temperatures (Q10 effect). The loss of the PBS antenna allows furthermore the attachment of recombinant proteins to the thylakoid membranes. The majority of enzymes are active at temperatures higher than 25°C. As a soil-dwelling organism the green alga Chlamydomonas reinhardtii, for example, is rarely exposed to high temperatures. The temperature optimum of its hydrogenase, however, is at 60°C (Happe and Naber, 1993). In conclusion, our data reveal that C. merolae acclimates to suboptimal growth temperature by remodeling its photosynthetic apparatus, by tuning its photosynthetic capacity, and by adjusting its machinery for protein folding, degradation, and homeostasis. MATERIALS AND METHODS Cultivation of Cyanidioschyzon merolae The red algae Cyanidioschyzon merolae 10D was kindly provided by Professor Tsuneyoshi Kuroiwa (Department of Life Science, Rikkyo University, Tokyo, Japan). It was cultivated in 2× Allen’s medium as described by Minoda et al. (2004) at 42°C with 100 rpm shaking under continuous light of 70 µmol photons m−2 s−1. For studies on the influence of the temperature on the alga, one liter of the medium was inoculated with liquid 42°C culture and split into two 2-L cotton-plugged flasks and incubated at 25°C or 42°C. Culture inoculated with 25°C cells were termed long-term adapted 25°C culture. Every 72 h 1 mL of each culture was taken and employed for flow cytometry measurements. For all of the performed experiments, exponentially growing cells were employed. For isotopic 15N labeling, the exponentially growing 42°C cells had to be diluted in 15N medium at least once. All other experiments were performed on cultures in the exponential growth phase (approximately 1 month after inoculation). Flow Cytometry Growth of the C. merolae cultures at 25°C and 42°C was monitored by means of a flow cytometer (BD FACS Canto II) on two biological replicates in two independent experiments (Fig. 1; Supplemental Fig. S1). Laser voltage was set as follows: forward-scatter (FSC), 645; side-scatter, 480; fluorescein isothiocyanate, 280; Pacific blue, 620; Allophycocyanin, 290; PerCP-Cy5-5, 374. Fifty thousand events (cells and bead reference) were recorded at a low to medium flow rate, identified by the FSC and side-scatter characteristics values, acquired in linear mode. For discrimination of living from dead cells, the fluorescent dye DAPI was employed, whose fluorescence was detected by the Pacific blue signal. Five microliters of the culture were mixed with 2× Allen’s medium and DAPI at a final concentration of 2 mg L−1 and Alexa Fluor 488-labeled standard particles solution (Polyscience, 4 mm microspheres), adjusted to a concentration of 1.2*105 mL−1 (30,000 standard particles in the mixture). Chlorophyll fluorescence was followed using the PerCP-Cy5-5 filter settings (excitation at 488 nm, detection at 695 nm). Phycocyanin fluorescence was measured using the Allophycocyanin filter settings (excitation at 633 nm, detection at 660 nm). Fluorescence intensities were acquired at log scale. All flow cytometry data were analyzed with the software BD FACSDiva, Version v6.12. Cellular debris with low FSC characteristics and dead cells (DAPI-positive) were excluded from further evaluation. Absolute numbers of 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 a Clark-type oxygen electrode as described in Naumann et al. (2007). The electrode was calibrated using dithionite. Samples were dark incubated for 20 min either at 25°C or 42°C before each measurement, which was carried out at 42°C in the dark or at different light intensities (from 50 to 500 µmol photons m−2 s−1). Protein Separation and Immunoblot Analysis Whole-cell samples (50 mg total protein, measured by Pierce BCA Protein Assay Kit) were analyzed by discontinuous 13% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) and either stained with Coomassie Brilliant Blue or transferred 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 phycocyanin alpha (CPCA) and beta (CPCB) chain (1: 5,000 dilution). After following incubation with an anti-rabbit peroxidase-conjugated antibody, the signals were detected by enhanced chemical luminescence. After staining, single SDS-PAGE bands were excised and tryptic in-gel digested, prior to mass spectrometric analysis. Isolation of Thylakoid Membranes Thylakoid membranes of 25°C and 42°C C. merolae cultures (14N as well as 15N labeled) were isolated as described previously (Chua and Bennoun, 1975; Petroutsos et al., 2009), breaking the cells by passing them two times through a self-made bio-nebulizer at a pressure of 20 psi. The thylakoids were either treated with NaBr (+NaBr samples) or not (−NaBr samples) and solubilized with n-dodecyl b-d maltoside prior to an ultracentrifugation on a linear Suc gradient (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, pH 7.5, 5 mm MgCl2, 10 mm NaCl) to a concentration of 2 mg ml−1 chlorophyll, and mixed with NaBr (2 m final concentration). After incubation on ice for 30 min, the mixture was diluted 1:2 with the same buffer and centrifuged at 10,000g, 4°C for 10 min. The resulting pellet was resuspended in 5 mm HEPES, pH 7.5, 10 mm EDTA and washed twice. SDG Ultracentrifugation Solubilized thylakoid membranes (0.8 mg ml−1 chlorophyll) from 25°C and 42°C cultures were loaded on separate discontinuous Suc density gradients with Suc concentrations from 1.3 to 0.1 m (0.05% β-DM) and ultracentrifuged at 33,000 rpm for 14 to 16 h using an SW41Ti rotor (Beckmann) at 4°C (Takahashi et al., 2006). SDGs were fractionated from bottom to top (approximately 300 µL per fraction). Low-Temperature (77 K) Fluorescence Emission 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 fluorescence emission spectra were recorded with the FP-6500 spectrofluorometer (Jasco). The samples were excited at 435 nm for chlorophyll a or 600 nm for phycocyanin, and the fluorescence emission was recorded from 600 to 800 nm or 620 to 800 nm, respectively. All spectra were normalized to the PSI signal at 728 nm. FASP of SDG Fractions Protein concentration of each SDG fraction was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions. For label-free quantification, the same volume of each fraction was loaded on separate Amicon Ultra 0.5 mL centrifugal filters (30-kD cutoff; Millipore) according to the FASP method (Wiśniewski et al., 2009, 2011) with minor modifications. Same volume of each 14N and 15N fraction was tryptically digested in one filter device. Proteins were reduced with 100 mm dithiothreitol in 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. Alkylation of Cys residues was performed by adding 50 mm iodoacetamide in UA followed by incubation for 20 min in the dark. Afterward, samples were washed each three times with UA and 50 mm NH4HCO3. Following tryptical digestion overnight (enzyme-to-protein ratio 1:50), peptides were eluted from the filter by centrifugation, acidified with 20 µL of 2% (v/v) formic acid, and dried by vacuum centrifugation. LC-MS/MS and Data Analysis Whole Cells Whole-cell extracts of exponentially growing C. merolae cultures (25°C versus 42°C) were mixed on equal protein amount [50 µg from each condition; the 42°C culture was isotopically labeled with heavy nitrogen (15N)] and separated by SDS-PAGE. Each gel lane was cut into up to 50 bands. Gel slices were in-gel-digested using trypsin, and resulting peptides were subjected to liquid chromatography coupled with high-resolution mass spectrometry. Only proteins identified and quantified in all three sets were further analyzed. Three different protein sets were analyzed: protein extracts from 42°C culture mixed with 25°C culture switched from 42°C (set1), a second biological replicate of the same conditions (set3), and a mix of 42°C culture with long-term adapted 25°C culture (set2). MS analysis, peptide identification (using Uniprot reference proteome database of C. merolae, ProteomeId UP000007014), determination of false discovery rates, and protein quantification were performed as described by 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 replicate), as well as on 14N-labeled 42°C versus 15N-labeled 25°C fractions (second biological replicate). Additionally, the +NaBr as well as the −NaBr samples from both biological replicates were mass spectrometrically analyzed. Liquid chromatography was performed on an Ultimate 3000 nanoRSLC system (Thermo Scientfic) coupled via a nanospray interface to a Q Exactive Plus mass spectrometer (Thermo Scientific). An estimated 2 µg of peptides were loaded on a trap column (C18 PepMap 100, 300 µm × 5 mm, 5 µm particle size, 100 Å pore size; Thermo Scientific) using 0.05% (v/v) trifluoroacetic acid/2% (v/v) acetonitrile and desalted for 2 min at a flow rate of 40 µL/min. For peptide separation, an Acclaim Pepmap capillary column (75 µm × 15 cm, 2 µm particle size, 100 Å pore size; Thermo Scientific) was used. Mobile phases consisted of 0.1% (v/v) formic acid in ultrapure water (A) and 0.1% (v/v) formic acid/80% (v/v) acetonitrile in ultrapure water (B). Gradient elution was carried out as follows: 2.5% to 30% B over 93 min, 30% to 50% over 7 min, 50% to 99% B over 3 min, 99% B over 10 min. The mass spectrometer was operated in a data-dependent mode that automatically switched between one survey scan (m/z 375–1400, resolution 70,000 at m/z 200, automatic gain control target value 1e6, maximum injection time 30 ms) and up to 12 higher-energy C-trap dissociation fragmentation scans on the 12 most intense ions (27% normalized collision energy, resolution 17,500 at m/z 200, automatic gain control target value 1e5, underfill ratio 1%, maximum injection time 64 ms, dynamic exclusion 20 s, precursor isolation window 1.5 m/z). Label-Free Quantification MS data were searched against the Uniprot reference proteome database of C. merolae (ProteomeId UP000007014) using MaxQuant (Version 1.5.3.30; Cox and Mann, 2008). Default search and quantification settings were applied with the following exceptions: “match between runs” was activated and protein decoy sequences were generated by randomization. Due to the heterogenous nature of SDG fractions, nonnormalized protein intensities instead of label-free quantification intensities were used for the preparation of figures. Quantification of 14N/15N-Labeled Proteins MS raw files were converted to mzML format with msconvert (Proteowizard version 3.0.7692; Kessner et al., 2008). Peptide identification and quantification was carried out essentially as described for labeled whole cells. Instead of computing 14N/15N intensity ratios, protein intensities for each labeling state were calculated by summing up peptide intensities. MS data have been deposited to ProteomeXchange via the PRIDE partner repository (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 of C. merolae 25°C and 42°C cultures (second biological replicate). Supplemental Figure S2. Low-temperature (77 K) fluorescence emission spectroscopy 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 intensities throughout the SDGs of the label-free 25°C and 42°C sample. Supplemental Table S1. Additional ratios of proteins identified in two of the three quantified protein data sets. Glossary PBS phycobilisomes LHC light-harvesting complex LITERATURE CITED Adir N ( 2005 ) Elucidation of the molecular structures of components of the phycobilisome: Reconstructing a giant . Photosynth Res 85 : 15 – 32 Google Scholar Crossref Search ADS PubMed WorldCat Allen MM ( 1984 ) Cyanobacterial cell inclusions . Annu Rev Microbiol 38 : 1 – 25 Google Scholar Crossref Search ADS PubMed WorldCat Atkin OK , Tjoelker MG ( 2003 ) Thermal acclimation and the dynamic response of plant respiration to temperature . Trends Plant Sci 8 : 343 – 351 Google Scholar Crossref Search ADS PubMed WorldCat Bruce D , Biggins J, Steiner T, Thewalt M ( 1985 ) Mechanism of the light state transition in photosynthesis. 4. Picosecond fluorescence spectroscopy of Anacystis nidulans and Porphyridium cruentum in state 1 and state 2 at 77 K . Biochim Biophys Acta 806 : 237 – 246 Google Scholar Crossref Search ADS WorldCat Busch A , Nield J, Hippler M ( 2010 ) The composition and structure of photosystem I-associated antenna from Cyanidioschyzon merolae . Plant J 62 : 886 – 897 Google Scholar Crossref Search ADS PubMed WorldCat Chua NH , Bennoun P ( 1975 ) Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: Wild-type and mutant strains deficient in photosystem II reaction center . Proc Natl Acad Sci USA 72 : 2175 – 2179 Google Scholar Crossref Search ADS PubMed WorldCat Chukhutsina V , Bersanini L, Aro EM, van Amerongen H ( 2015 ) Cyanobacterial light-harvesting phycobilisomes uncouple from photosystem I during dark-to-light transitions . Sci Rep 5 : 14193 Google Scholar Crossref Search ADS PubMed WorldCat Ciniglia C , Yoon HS, Pollio A, Pinto G, Bhattacharya D ( 2004 ) Hidden biodiversity of the extremophilic Cyanidiales red algae . Mol Ecol 13 : 1827 – 1838 Google Scholar Crossref Search ADS PubMed WorldCat Cox J , Mann M ( 2008 ) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification . Nat Biotechnol 26 : 1367 – 1372 Google Scholar Crossref Search ADS PubMed WorldCat Doemel WN , Brock TD ( 1971 ) The physiological ecology of Cyanidium caldarium . J Gen Microbiol 67 : 17 – 32 Google Scholar Crossref Search ADS WorldCat Ferrer M , Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN ( 2003 ) Chaperonins govern growth of Escherichia coli at low temperatures . Nat Biotechnol 21 : 1266 – 1267 Google Scholar Crossref Search ADS PubMed WorldCat Geer LY , Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, Yang X, Shi W, Bryant SH ( 2004 ) Open mass spectrometry search algorithm . J Proteome Res 3 : 958 – 964 Google Scholar Crossref Search ADS PubMed WorldCat Görl M , Sauer J, Baier T, Forchhammer K ( 1998 ) Nitrogen-starvation-induced chlorosis in Synechococcus PCC 7942: Adaptation to long-term survival . Microbiology 144 : 2449 – 2458 Google Scholar Crossref Search ADS PubMed WorldCat Grossman AR , Schaefer MR, Chiang GG, Collier JL ( 1993 ) The phycobilisome, a light-harvesting complex responsive to environmental conditions . Microbiol Rev 57 : 725 – 749 Google Scholar Crossref Search ADS PubMed WorldCat Happe T , Naber JD ( 1993 ) Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii . Eur J Biochem 214 : 475 – 481 Google Scholar Crossref Search ADS PubMed WorldCat Hippler M , Drepper F, Farah J, Rochaix JD ( 1997 ) Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF . Biochemistry 36 : 6343 – 6349 Google Scholar Crossref Search ADS PubMed WorldCat Hoefnagel MHN , Atkin OK, Wiskich JT ( 1998 ) Interdependence between chloroplasts and mitochondria in the light and the dark . Biochim Biophys Acta 1366 : 235 – 255 Google Scholar Crossref Search ADS WorldCat Huner NPA , Öquist G, Sarhan F ( 1998 ) Energy balance and acclimation to light and cold . Trends Plant Sci 3 : 224 – 230 Google Scholar Crossref Search ADS WorldCat Kaňa R , Kotabová E, Luke¡ M, Papáček S, Matonoha C, Liu LN, Prá¡il O, Mullineaux CW ( 2014 ) Phycobilisome mobility and its role in the regulation of light harvesting in red algae . Plant Physiol 165 : 1618 – 1631 Google Scholar Crossref Search ADS PubMed WorldCat Kessner D , Chambers M, Burke R, Agus D, Mallick P ( 2008 ) ProteoWizard: Open source software for rapid proteomics tools development . Bioinformatics 24 : 2534 – 2536 Google Scholar Crossref Search ADS PubMed WorldCat Kirilovsky D ( 2015 ) Modulating energy arriving at photochemical reaction centers: Orange carotenoid protein-related photoprotection and state transitions . Photosynth Res 126 : 3 – 17 Google Scholar Crossref Search ADS PubMed WorldCat Kiseleva LL , Horvàth I, Vigh L, Los DA ( 1999 ) Temperature-induced specifc lipid desaturation in the thermophilic cyanobacterium Synechococcus vulcanus . FEMS Microbiol Lett 175 : 179 – 183 Google Scholar Crossref Search ADS WorldCat Kobayashi Y , Harada N, Nishimura Y, Saito T, Nakamura M, Fujiwara T, Kuroiwa T, Misumi O ( 2014 ) Algae sense exact temperatures: Small heat shock proteins are expressed at the survival threshold temperature in Cyanidioschyzon merolae and Chlamydomonas reinhardtii . Genome Biol Evol 6 : 2731 – 2740 Google Scholar Crossref Search ADS PubMed WorldCat Kondo K , Ochiai Y, Katayama M, Ikeuchi M ( 2007 ) The membrane-associated CpcG2-phycobilisome in Synechocystis: A new photosystem I antenna . Plant Physiol 144 : 1200 – 1210 Google Scholar Crossref Search ADS PubMed WorldCat Krupnik T , Kotabová E, van Bezouwen LS, Mazur R, Garstka M, Nixon PJ, Barber J, Kaňa R, Boekema EJ, Kargul J ( 2013 ) A reaction center-dependent photoprotection mechanism in a highly robust photosystem II from an extremophilic red alga, Cyanidioschyzon merolae . J Biol Chem 288 : 23529 – 23542 Google Scholar Crossref Search ADS PubMed WorldCat Kuroiwa T , Kawazu T, Takahashi H, Suzuki K, Ohta N, Kuroiwa H ( 1994 ) Comparison of ultrastructures between the ultra-small eukaryote Cyanidioschyzon merolae and Cyanidium caldarium . Cytologia (Tokyo) 59 : 149 – 158 Google Scholar Crossref Search ADS WorldCat Laemmli UK ( 1970 ) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227 : 680 – 685 Google Scholar Crossref Search ADS PubMed WorldCat Liu H , Zhang H, Niedzwiedzki DM, Prado M, He G, Gross ML, Blankenship RE ( 2013 ) Phycobilisomes supply excitations to both photosystems in a megacomplex in cyanobacteria . Science 342 : 1104 – 1107 Google Scholar Crossref Search ADS PubMed WorldCat Matsuzaki M , Misumi O, Shin-I T, Maruyama S, Takahara M, Miyagishima SY, Mori T, Nishida K, Yagisawa F, Nishida K, et al. ( 2004 ) Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D . Nature 428 : 653 – 657 Google Scholar Crossref Search ADS PubMed WorldCat Merola A , Castaldo R, De Luca P, Gambardella R, Musachio A, Taddei R ( 1981 ) Revision of Cyanidium caldarium. Three species of acidophilic algae . Giorn Bot Ital. 115 : 189 – 195 Google Scholar Crossref Search ADS WorldCat Minoda A , Sakagami R, Yagisawa F, Kuroiwa T, Tanaka K ( 2004 ) Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D . Plant Cell Physiol 45 : 667 – 671 Google Scholar Crossref Search ADS PubMed WorldCat Murata N , Los DA ( 1997 ) Membrane fluidity and temperature perception . Plant Physiol 115 : 875 – 879 Google Scholar Crossref Search ADS PubMed WorldCat Naumann B , Busch A, Allmer J, Ostendorf E, Zeller M, Kirchhoff H, Hippler M ( 2007 ) Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii . Proteomics 7 : 3964 – 3979 Google Scholar Crossref Search ADS PubMed WorldCat Neilson JA , Durnford DG ( 2010 ) Structural and functional diversification of the light-harvesting complexes in photosynthetic eukaryotes . Photosynth Res 106 : 57 – 71 Google Scholar Crossref Search ADS PubMed WorldCat Nozaki H , Takano H, Misumi O, Terasawa K, Matsuzaki M, Maruyama S, Nishida K, Yagisawa F, Yoshida Y, Fujiwara T, et al. ( 2007 ) A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae . BMC Biol 5 : 28 Google Scholar Crossref Search ADS PubMed WorldCat Ohta N , Matsuzaki M, Misumi O, Miyagishima SY, Nozaki H, Tanaka K, Shin-I T, Kohara Y, Kuroiwa T ( 2003 ) Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae . DNA Res 10 : 67 – 77 Google Scholar Crossref Search ADS PubMed WorldCat Padmasree K , Padmavathi L, Raghavendra AS ( 2002 ) Essentiality of mitochondrial oxidative metabolism for photosynthesis: Optimization of carbon assimilation and protection against photoinhibition . Crit Rev Biochem Mol Biol 37 : 71 – 119 Google Scholar Crossref Search ADS PubMed WorldCat Petroutsos D , Terauchi AM, Busch A, Hirschmann I, Merchant SS, Finazzi G, Hippler M ( 2009 ) PGRL1 participates in iron-induced remodeling of the photosynthetic apparatus and in energy metabolism in Chlamydomonas reinhardtii . J Biol Chem 284 : 32770 – 32781 Google Scholar Crossref Search ADS PubMed WorldCat Reeb V , Bhattacharya D ( 2010 ) The thermos-acidophilic Cyanidiophyceae (Cyanidiales) . In J Seckbach , DJ Chapman , eds, Red Algae in the Genomic Age . Springer , Dordrecht, Netherlands , pp 409 – 426 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Richaud C , Zabulon G, Joder A, Thomas JC ( 2001 ) Nitrogen or sulfur starvation differentially affects phycobilisome degradation and expression of the nblA gene in Synechocystis strain PCC 6803 . J Bacteriol 183 : 2989 – 2994 Google Scholar Crossref Search ADS PubMed WorldCat Shen G , Boussiba S, Vermaas WF ( 1993 ) Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function . Plant Cell 5 : 1853 – 1863 Google Scholar PubMed OpenURL Placeholder Text WorldCat Stadnichuk IN , Bulychev AA, Lukashev EP, Sinetova MP, Khristin MS, Johnson MP, Ruban AV ( 2011 ) Far-red light-regulated efficient energy transfer from phycobilisomes to photosystem I in the red microalga Galdieria sulphuraria and photosystems-related heterogeneity of phycobilisome population . Biochim Biophys Acta 1807 : 227 – 235 Google Scholar Crossref Search ADS PubMed WorldCat Su X , Fraenkel PG, Bogorad L ( 1992 ) Excitation energy transfer from phycocyanin to chlorophyll in an apcA-defective mutant of Synechocystis sp. PCC 6803 . J Biol Chem 267 : 22944 – 22950 Google Scholar Crossref Search ADS PubMed WorldCat Takahashi H , Iwai M, Takahashi Y, Minagawa J ( 2006 ) Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii . Proc Natl Acad Sci USA 103 : 477 – 482 Google Scholar Crossref Search ADS PubMed WorldCat Terashima M , Specht M, Naumann B, Hippler M ( 2010 ) Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics . Mol Cell Proteomics 9 : 1514 – 1532 Google Scholar Crossref Search ADS PubMed WorldCat Vizcaíno JA , Csordas A, del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez-Riverol Y, Reisinger F, Ternent T, et al. ( 2016 ) 2016 update of the PRIDE database and its related tools . Nucleic Acids Res 44 ( D1 ): D447 – D456 Google Scholar Crossref Search ADS PubMed WorldCat Wiemann P , Sieber CMK, von Bargen KW, Studt L, Niehaus EM, Espino JJ, Huß K, Michielse CB, Albermann S, Wagner D, et al. ( 2013 ) Deciphering the cryptic genome: Genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites . PLoS Pathog 9 : e1003475 Google Scholar Crossref Search ADS PubMed WorldCat Wiśniewski JR , Zielinska DF, Mann M ( 2011 ) Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method . Anal Biochem 410 : 307 – 309 Google Scholar Crossref Search ADS PubMed WorldCat Wiśniewski JR , Zougman A, Nagaraj N, Mann M ( 2009 ) Universal sample preparation method for proteome analysis . Nat Methods 6 : 359 – 362 Google Scholar Crossref Search ADS PubMed WorldCat Wolfe GR , Cunningham FX Jr , Durnford D, Green BR, Gantt E ( 1994 ) Evidence for a common origin of chloroplasts with light-harvesting complexes of different pigmentation . Nature 367 : 566 – 568 Google Scholar Crossref Search ADS WorldCat Yoon HS , Hackett JD, Ciniglia C, Pinto G, Bhattacharya D ( 2004 ) A molecular timeline for the origin of photosynthetic eukaryotes . Mol Biol Evol 21 : 809 – 818 Google Scholar Crossref Search ADS PubMed WorldCat Yoon HS , Müller KM, Sheath RG, Ott FD, Bhattacharya D ( 2006 ) Defining the major lineages of red algae (rhodophyta) . J Phycol 42 : 482 – 492 Google Scholar Crossref Search ADS WorldCat Author notes 1 This work was supported by the Deutsche Forschungsgemeinschaft (HI 739/13-1 to M.H.). 2 These authors contributed equally to the article. 3 Present address: Institute of Applied Microbiology, Aachen Biology and Biotechnology, RWTH Aachen University, 52074 Aachen, Germany. * Address correspondence to mhippler@uni-muenster.de. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michael Hippler (mhippler@uni-muenster.de). D.W. and M.H. designed the research; D.W. and D.N performed the 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 scripts for analysis of proteomics data; D.N., D.W., and M.H. wrote the manuscript. www.plantphysiol.org/cgi/doi/10.1104/pp.17.00110 © 2017 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Temperature-Induced Remodeling of the Photosynthetic Machinery Tunes Photosynthesis in the Thermophilic Alga Cyanidioschyzon merolae  
JF - Plant Physiology
DO - 10.1104/pp.17.00110
DA - 2017-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/temperature-induced-remodeling-of-the-photosynthetic-machinery-tunes-Of0U48b9ZH
SP - 35
EP - 46
VL - 174
IS - 1
DP - DeepDyve
ER -