TY - JOUR
AU - Hüner, Norman P.A.
AB - Abstract Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b 6/f (Cyt b 6/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b 6/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700+ indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b 6/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b 6/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins. The Antarctic psychrophilic green alga Chlamydomonas sp. UWO 241 (UWO 241) originates from the lowest trophic zone of Lake Bonney, which is characterized by an extremely stable environment of low temperatures (4°C–6°C), low irradiance (less than 50 µmol photons m−2 s−1), high salt concentrations (700 mm), and a narrow spectral distribution enriched in the blue-green region (Lizotte and Priscu, 1992; Morgan-Kiss et al., 2006). Adaptation of UWO 241 to this unique natural aquatic environment has resulted in the evolution of a structurally and functionally distinct photosynthetic apparatus relative to the mesophilic strains Chlamydomonas raudensis SAG 49.72 (SAG 49.72; Pocock et al., 2004) and the model green alga Chlamydomonas reinhardtii (Morgan et al., 1998; Morgan-Kiss et al., 2006). UWO 241 is a halotolerant psychrophile (Morgan-Kiss et al., 2006; Takizawa et al., 2009) that dies at temperatures of 20°C or higher (Possmayer et al., 2011). This is consistent with the fact that temperature-response curves for light-saturated rates of CO2-saturated oxygen evolution indicate that UWO 241 photosynthesizes maximally at 8°C at rates that are comparable to rates of the mesophile, C. reinhardtii, grown and measured at 29°C (Pocock et al., 2007). Although UWO 241 exhibits a low quantum requirement for photoinhibition and the degradation of the PSII reaction center polypeptide D1 (PsbA), this is complemented by a rapid, light-dependent recovery of PSII photochemistry associated with the de novo biosynthesis of D1 at low temperature (Pocock et al., 2007). Thus, this psychrophile appears to be photosynthetically adapted to growth at low temperature (Pocock et al., 2007). UWO 241 exhibits significantly enhanced fatty acid unsaturation associated with all of the major thylakoid lipid classes (monogalactosyldiacylglyceride, digalactosyldiacylglyceride, sulfoquinovosyldiacylglyceride, and phosphatidyldiacylglyceride) as well as a 2- to 10-fold increase in the unique, unsaturated fatty acid 16:4, depending on the specific thylakoid lipid species (Morgan-Kiss et al., 2002a). Consequently, the biophysical determination of the critical temperature for thylakoid membrane destabilization for UWO 241 (40°C) was significantly lower than that for C. reinhardtii (50°C), which is consistent with the adaptation of UWO 241 to low temperature (Morgan-Kiss et al., 2002a). Biochemical analyses of the chlorophyll-protein complexes coupled with immunoblots of their constituent polypeptides indicate that UWO 241 exhibits abundant PSII light-harvesting complex (LHCII) associated with a low chlorophyll a/b (Chl a/b) ratio (1.8–2) relative to the mesophiles, SAG 49.72 and C. reinhardtii (Chl a/b ratio = 3). In addition, UWO 241 exhibits an unusually low level of PSI such that the stoichiometry of PSI/PSII was estimated to be about 0.5 in UWO 241, whereas the mesophiles, SAG 49.72 and C. reinhardtii, grown under optimal growth conditions, exhibited a PSI/PSII of about 1. These biochemical data were confirmed by measurements of P700 photooxidation (Morgan-Kiss et al., 2002b; Szyszka et al., 2007), which indicated that UWO 241 exhibits high rates of PSI cyclic electron flow (CEF; Morgan-Kiss et al., 2002b). Recently, we reported that acclimation of UWO 241 to low temperature and low growth irradiance results in alterations in the partitioning of excess excitation energy to maintain cellular energy balance compared with the mesophile, SAG 49.72 (Szyszka et al., 2007). While SAG 49.72 favors energy partitioning for photoprotection through the induction of the xanthophyll cycle, the psychrophilic strain, UWO 241, favors energy partitioning for photoprotection through constitutive quenching processes involved in energy dissipation, even though UWO 241 exhibits an active xanthophyll cycle (Pocock et al., 2007; Szyszka et al., 2007). Although the molecular basis of the constitutive quenching process for photoprotection has not been elucidated unequivocally, this may reflect the differences in the predisposition for energy dissipation through either the Q2 or the Q1 site in PSII-LHCII supercomplexes (Jahns and Holzwarth 2012; Derks et al., 2015) or, alternatively, it may indicate quenching through PSII reaction centers, as suggested previously (Hüner et al., 2006; Sane et al., 2012). Regardless of the mechanism, one consequence of this enhanced energy-quenching capacity of UWO 241 is that the psychrophile does not exhibit any pigment change in response to photoacclimation (Morgan-Kiss et al., 2006), typically observed for other mesophilic green algae such as C. reinhardtii, Dunaliella tertiolecta (Escoubas et al., 1995), Dunaliella salina (Smith et al., 1990; Maxwell et al., 1995), and Chlorella vulgaris (Maxwell et al., 1995; Wilson et al., 2003). In addition, maximum growth rates of UWO 241 are sensitive to light quality, since rates of growth and photosynthesis are inhibited under red light, which results in increased excitation pressure in the psychrophile (Morgan-Kiss et al., 2005). However, the most unusual feature of UWO 241 is that it represents a natural variant that is deficient in state transitions (Morgan-Kiss et al., 2002b; Takizawa et al., 2009). State transitions have been well documented as a short-term mechanism for photoacclimation employed by algae and plants to balance light excitation between PSII and PSI (Allen et al., 1981; Allen, 2003; Eberhard et al., 2008; Rochaix, 2011, 2014). Overexcitation of PSII relative to PSI results in the phosphorylation of several peripheral Chl a/b-binding LHCII proteins, which causes their dissociation from the PSII core and subsequent association with PSI (Eberhard et al., 2008; Rochaix, 2011). As a result, excitation energy is redistributed in favor of PSI at the expense of PSII. Phosphorylation of LHCII polypeptides is essential in the regulation of state transitions and energy distribution between the two photosystems (Allen, 2003; Eberhard et al., 2008; Kargul and Barber, 2008; Rochaix, 2011, 2014). LHCII phosphorylation is initiated by modulation of the redox state of the plastoquinone (PQ) pool, which is sensed through the preferential binding of plastoquinol to the quinone-binding site of the cytochrome b 6/f (Cyt b 6/f) complex. As a consequence, the thylakoid protein kinases STT7 in C. reinhardtii and its ortholog, STN7, in Arabidopsis (Arabidopsis thaliana) are activated and LHCII is phosphorylated (Rochaix, 2011, 2014; Wunder et al., 2013). Similar to all other photosynthetic organisms, the LHCII polypeptides represent the major phosphorylated polypeptides detected in thylakoids of the mesophile, SAG 49.72 (Szyszka et al., 2007). Consistent with a deficiency in state transitions, UWO 241 does not phosphorylate the major LHCII polypeptides in response to changes in either growth irradiance or growth temperature (Morgan-Kiss et al., 2002b; Szyszka et al., 2007; Takizawa et al., 2009). In fact, UWO 241 exhibits a unique thylakoid membrane phosphorylation profile compared with either SAG 49.72 or C. reinhardtii (Morgan-Kiss et al., 2005; Szyszka et al., 2007; Takizawa et al., 2009). Rather than phosphorylation of LHCII polypeptides, UWO 241 preferentially phosphorylates several novel high-molecular-mass polypeptides (greater than 70 kD; Morgan-Kiss et al., 2002b; Szyszka et al., 2007). The Cyt b 6/f complex of the photosynthetic intersystem electron transport chain is essential in the regulation of state transitions and the activation of the STT7 kinase (Rochaix, 2011, 2014). The Cyt b 6/f complex of UWO 241 exhibits a unique cytochrome f (Cyt f) that is 7 kD smaller than the expected molecular mass of 41 kD exhibited by C. reinhardtii based on SDS-PAGE (Morgan-Kiss et al., 2006; Gudynaite-Savitch et al., 2006, 2007). No other differences in the structure and composition of the Cyt b 6/f complex are apparent. Sequencing of the entire Cytochrome f gene (petA) from UWO 241 indicated that the amino acid sequence of Cyt f from UWO 241 exhibited 79% identity to that of C. reinhardtii. Through domain swapping between petA of UWO 241 and that of C. reinhardtii and subsequent transformation of a ƊpetA mutant of C. reinhardtii with the chimeric gene constructs, we reported that the apparent differences in molecular masses observed for petA in UWO 241 are due to differences in the amino acid sequences of the small domain of Cyt f. However, complementation of the ƊpetA mutant of C. reinhardtii with the entire petA from either UWO 241 or C. reinhardtii completely restored the capacity for state transitions in the ƊpetA mutant. Thus, we concluded that the changes in the amino acid sequence of the small domain of Cyt f of UWO 241 cannot account for the inability of UWO 241 to undergo state transitions (Gudynaite-Savitch et al., 2006, 2007). Since state transitions are inhibited in UWO 241, we hypothesized that the unique protein phosphorylation pattern observed in UWO 241 reflects an alternative mechanism to regulate energy flow within the photosynthetic apparatus of this Antarctic psychrophile. Thus, the objective of this research was to identify and characterize the high-molecular-mass polypeptides phosphorylated in the psychrophile, UWO 241. We report that UWO 241 preferentially phosphorylates specific polypeptides associated with a PSI-Cyt b 6/f supercomplex. The role of the PSI-Cyt b 6/f supercomplex and its phosphorylation status in the regulation of PSI cyclic electron transport in UWO 241 are discussed. We suggest that adaptation of UWO 241 to its unique low-temperature and low-light environment favors the phosphorylation of a PSI-Cyt b 6/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of LHCII proteins. RESULTS Two-Dimensional Blue-Native Gel Electrophoresis of Thylakoid Protein Complexes Figure 1 indicates that the mesophile, SAG 49.72, and the psychrophile, UWO 241, exhibited comparable levels of LCHII polypeptides (Fig. 1A, arrow). Consistent with previously published data (Szyszka et al., 2007), SAG 49.72 exhibited the expected phosphorylation of LHCII, the core PSII complex CP43, as well as the reaction center polypeptide D1 (Fig. 1B). In contrast, the psychrophile, UWO 241, exhibited minimal phosphorylation of these PSII and LHCII polypeptides but rather exhibited major thylakoid protein phosphorylation bands at a molecular mass greater than 130 kD as well as a polypeptide of about 17 kD (Fig. 1B, arrows). As an initial step in the identification and characterization of these novel phosphorylated thylakoid protein(s) in UWO 241, we used two-dimensional nondenaturing blue-native PAGE (BN-PAGE), which allowed for the separation of more than 50 different polypeptide subunits that represent components of thylakoid multisubunit complexes (Kugler et al., 1997). Figure 1. Open in new tabDownload slide Phosphorylation profiles of UWO 241 and SAG 49.72. A, Thylakoid membrane proteins (the arrow indicates major LHCII polypeptides) were isolated in the presence of 20 mm NaF, separated by SDS-PAGE, and stained with Coomassie Blue. B, Corresponding membranes were probed with antibodies specific for phospho-Thr. Arrows indicate the novel, high-molecular-mass protein phosphorylated band in UWO 241. Apparent molecular masses are indicated on the left (kilodaltons). Figure 1. Open in new tabDownload slide Phosphorylation profiles of UWO 241 and SAG 49.72. A, Thylakoid membrane proteins (the arrow indicates major LHCII polypeptides) were isolated in the presence of 20 mm NaF, separated by SDS-PAGE, and stained with Coomassie Blue. B, Corresponding membranes were probed with antibodies specific for phospho-Thr. Arrows indicate the novel, high-molecular-mass protein phosphorylated band in UWO 241. Apparent molecular masses are indicated on the left (kilodaltons). Thylakoid membranes from SAG 49.72 (Fig. 2A, lanes 1 and 2) and UWO 241 (Fig. 2A, lanes 3 and 4) were solubilized in the presence of β-dodecyl maltoside, and the resultant intact protein complexes were separated in the first dimension on a blue-native gel according to their molecular mass. Although both SAG 49.72 and UWO 241 exhibited the presence of nine thylakoid protein complexes of molecular masses that varied between 150 and 750 kD, the relative abundances of these protein complexes varied dramatically (Fig. 2A). To identify these protein complexes, the first dimension was subjected to SDS-PAGE and subsequently probed with specific antibodies to major thylakoid protein complexes, which allowed us to identify most of the major complexes separated in the first dimension (Supplemental Fig. S1). Separation of thylakoid complexes by blue-native gel electrophoresis revealed several differences between the major thylakoid membrane complexes of SAG 49.72 and UWO 241. Consistent with previous results (Morgan et al., 1998), UWO 241 exhibited a decreased abundance of PSI+LHCI complexes, which migrate at approximately 700 kD (Fig. 2A). The majority of the LHCII complexes from the mesophilic strain migrated as a band of lower apparent molecular mass of approximately 240 kD, compared with the major LHCII band of the psychrophile, which appeared at approximately 300 kD, suggesting that SAG 49.72 and UWO 241 exhibit altered LHCII structure and/or protein conformation. In addition, the ATPase, PSI, and PSII core complexes in UWO 241 exhibited reduced binding of the stain, G250, relative to those present in SAG 49.72 (Fig. 2A). Figure 2. Open in new tabDownload slide BN-PAGE of thylakoid membrane complexes and subsequent analysis of SDS-solubilized complexes. A, Purified thylakoids of SAG 49.72 and UWO 241 were solubilized with 1% (w/v) n-dodecyl-β-maltoside (DDM), loaded on an equal chlorophyll (50 μg) basis, and separated on a 3% to 16% gradient native gel. B and C, BN-PAGE gel strips from SAG 49.72 and UWO 241 were treated with 1% (w/v) SDS and β-mercaptoethanol, and denatured subunits were separated using SDS-PAGE. D and E, Second dimension gels were stained with Coomassie Blue. Corresponding membranes were immunoblotted with phospho-Thr antibodies. Molecular masses are indicated (kilodaltons). Figure 2. Open in new tabDownload slide BN-PAGE of thylakoid membrane complexes and subsequent analysis of SDS-solubilized complexes. A, Purified thylakoids of SAG 49.72 and UWO 241 were solubilized with 1% (w/v) n-dodecyl-β-maltoside (DDM), loaded on an equal chlorophyll (50 μg) basis, and separated on a 3% to 16% gradient native gel. B and C, BN-PAGE gel strips from SAG 49.72 and UWO 241 were treated with 1% (w/v) SDS and β-mercaptoethanol, and denatured subunits were separated using SDS-PAGE. D and E, Second dimension gels were stained with Coomassie Blue. Corresponding membranes were immunoblotted with phospho-Thr antibodies. Molecular masses are indicated (kilodaltons). The second dimension SDS-PAGE gels (Fig. 2, B and C) indicated qualitative as well as quantitative differences in the polypeptide complement of the various thylakoid protein complexes present in SAG 49.72 (Fig. 2B) compared with UWO 241 (Fig. 2C). Subsequently, phospho-Thr antibodies were used to detect the phosphorylated polypeptides present in the intact membrane protein complexes of SAG 49.72 (Fig. 2D) and UWO 241 (Fig. 2E) separated in the first dimension. Immunoblot analysis of the second dimension indicated that SAG 49.72 exhibited a major phosphorylated polypeptide of 28 to 30 kD associated with LHCII and, hence, a component of the PSII complex (Fig. 2D, spot 3). In addition, two phosphorylated polypeptides of 43 and 32 kD (Fig. 2D, spots 1 and 2) were identified as the PSII reaction core antenna polypeptides CP43 (PsbC) and D1 (psbA), respectively. However, no phosphorylated polypeptides of molecular mass 130 kD or greater were detected in SAG 49.72 thylakoid membranes (Fig. 2D). Thus, as expected, the major sites of thylakoid protein phosphorylation in SAG 49.72 were the subunits of the PSII-LHCII supercomplex. Even though UWO 241 exhibited significant levels of the PSII subunits D1, CP43, and LHCII, the psychrophile exhibited minimal phosphorylation of any of these PSII subunits, which is consistent with the results presented in Figure 1. Instead, we detected the phosphorylation of a high-molecular-mass (greater than 130 kD) complex in UWO 241 (Fig. 2E, spot 1). These phosphoprotein(s) originated from a large complex that migrated with an apparent molecular mass of approximately 1,000 kD, which is slightly larger than that of the PSI+LHCI antenna complex (approximately 700 kD; Fig. 2C). In addition, a smaller, 17-kD phosphoprotein comigrated with this large complex (Fig. 2E, spot 3). Thus, phosphorylation in the mesophilic SAG 49.72 strain appeared to be associated with the PSII core complex and LHCII, while the psychrophile, UWO 241, exhibited phosphorylation of high-molecular-mass polypeptides (130 kD or greater). We hypothesized that the phosphoproteins detected in UWO 241 were components of a PSI-associated pigment-protein complex. Fractionation and Purification of Thylakoid Membrane Complexes To identify the specific subunits associated with the high-molecular-mass protein complex that undergo phosphorylation in UWO 241, Suc gradient centrifugation was performed in order to fractionate, isolate, and purify the major pigment-protein complexes from thylakoid membranes of the psychrophile, UWO 241, and the mesophile, SAG 49.72 (Fig. 3A). The model green alga, C. reinhardtii strain 1690 (1690), was used as an additional internal control for comparison (Fig. 3A). Suc density ultracentrifugation of purified thylakoid membranes treated with DDM resulted in the separation of four distinct but comparable green bands in the mesophiles, SAG 49.72 and 1690 (Fig. 3A). Although UWO 241 also generated four distinct green bands, the densities and the relative proportions of the green bands in UWO 241 differed significantly from those observed in either SAG 49.72 or 1690 (Fig. 3A). Figure 3. Open in new tabDownload slide Fractionation of major thylakoid membrane complexes of SAG 49.72, UWO 241, and 1690. A, Thylakoid membranes were purified in the presence of NaF, solubilized with 1% (w/v) DDM, loaded on a continuous Suc density gradient (1.3–0.1 m), and ultracentrifuged. SC represents a band of high-density supercomplexes. B, Absorption spectra of fractionated complexes following Suc density centrifugation. C and D, 77 K fluorescence emission spectra of intact thylakoid membranes of UWO 241 (C) and subsequent purified fractions of LHCII, PSII, PSI, and supercomplexes (D). Emission peak maxima are indicated above each peak (nm). Figure 3. Open in new tabDownload slide Fractionation of major thylakoid membrane complexes of SAG 49.72, UWO 241, and 1690. A, Thylakoid membranes were purified in the presence of NaF, solubilized with 1% (w/v) DDM, loaded on a continuous Suc density gradient (1.3–0.1 m), and ultracentrifuged. SC represents a band of high-density supercomplexes. B, Absorption spectra of fractionated complexes following Suc density centrifugation. C and D, 77 K fluorescence emission spectra of intact thylakoid membranes of UWO 241 (C) and subsequent purified fractions of LHCII, PSII, PSI, and supercomplexes (D). Emission peak maxima are indicated above each peak (nm). In all cases, band 1 was the major complex separated on the Suc gradient based on chlorophyll concentration. The absorption spectra (Fig. 3B) and Chl a/b ratios (Table I) indicated that band 1 was a Chl a/b pigment protein complex in UWO 241 as well as in SAG 49.72 and 1690 (Supplemental Fig. S2, A and B). The 77 K fluorescence emission spectra for band 1 exhibited a single emission maximum between 678 and 681 nm (Fig. 3D; Supplemental Fig. S2, E and F), which is consistent with the identification of band 1 as LHCII in all three samples (Krause and Weis, 1991). Band 2 exhibited a Chl a/b ratio between 6.99 and 8.57 (Table I) and absorption spectra consistent with a chlorophyll a (Chl a) pigment protein complex in SAG 49.72 and 1690 (Supplemental Fig. S2, A and B) as well as UWO 241 (Fig. 4B). The 77 K fluorescence emission spectra of band 2 exhibited an emission maximum at 685 nm and a shoulder at 695 nm, which is consistent with the identification of band 2 as the PSII core complex (Krause and Weis, 1991; Fig. 3D; Supplemental Fig. S2, E and F). Band 3 also exhibited a Chl a/b ratio of 5.29 to 8.43 (Table I) and absorption spectra consistent with a Chl a pigment-protein complex in all three strains examined (Fig. 3B; Supplemental Fig. S2, A and B). However, band 3 exhibited a single 77 K fluorescence emission maximum between 712 and 715 nm, which is consistent with the tentative identification of this band as a PSI complex in both SAG 49.72 and 1690 as well as the psychrophile (Krause and Weis, 1991; Fig. 3D; Supplemental Fig. S2, E and F). We note with interest that, as expected, a major PSI emission band between 706 and 708 nm was detected in purified thylakoids from SAG 49.72 and 1690 (Supplemental Fig. S2, C and D). However, no major PSI emission band at 707 nm was detected in purified thylakoids from UWO 241 (Fig. 3C), even though purified PSI exhibited a 77 K fluorescence emission maximum between 712 and 715 nm (Fig. 3D). This unique aspect of the fluorescence emission of PSI in UWO 241 was not an artifact of thylakoid isolation, since 77 K fluorescence emission spectra of intact cells also exhibit the absence of the PSI emission peak at 712 nm (Morgan-Kiss et al., 2002b; Takizawa et al., 2009). Chl a/b ratios of purified thylakoid membrane complexes from each algal strain Table I. Chl a/b ratios of purified thylakoid membrane complexes from each algal strain Individual complexes were isolated using Suc gradient fractionation. Values represent means ± sd. Complex . 1690 . SAG 49.72 . UWO 241 . Band 1 1.50 ± 0.04 1.94 ± 0.03 1.32 ± 0.12 Band 2 8.48 ± 0.09 8.57 ± 0.40 6.99 ± 0.56 Band 3 7.33 ± 0.52 8.43 ± 0.91 5.29 ± 0.39 Band 4 4.98 ± 0.45 8.05 ± 2.09 2.58 ± 0.33 Complex . 1690 . SAG 49.72 . UWO 241 . Band 1 1.50 ± 0.04 1.94 ± 0.03 1.32 ± 0.12 Band 2 8.48 ± 0.09 8.57 ± 0.40 6.99 ± 0.56 Band 3 7.33 ± 0.52 8.43 ± 0.91 5.29 ± 0.39 Band 4 4.98 ± 0.45 8.05 ± 2.09 2.58 ± 0.33 Open in new tab Table I. Chl a/b ratios of purified thylakoid membrane complexes from each algal strain Individual complexes were isolated using Suc gradient fractionation. Values represent means ± sd. Complex . 1690 . SAG 49.72 . UWO 241 . Band 1 1.50 ± 0.04 1.94 ± 0.03 1.32 ± 0.12 Band 2 8.48 ± 0.09 8.57 ± 0.40 6.99 ± 0.56 Band 3 7.33 ± 0.52 8.43 ± 0.91 5.29 ± 0.39 Band 4 4.98 ± 0.45 8.05 ± 2.09 2.58 ± 0.33 Complex . 1690 . SAG 49.72 . UWO 241 . Band 1 1.50 ± 0.04 1.94 ± 0.03 1.32 ± 0.12 Band 2 8.48 ± 0.09 8.57 ± 0.40 6.99 ± 0.56 Band 3 7.33 ± 0.52 8.43 ± 0.91 5.29 ± 0.39 Band 4 4.98 ± 0.45 8.05 ± 2.09 2.58 ± 0.33 Open in new tab Figure 4. Open in new tabDownload slide Immunoblot analysis of purified complexes from SAG 49.72, UWO 241, and 1690. A to C, Thylakoid membrane proteins and fractions isolated by Suc density centrifugation from SAG 49.72 (A), 1690 (B), and UWO 241 (C) were Coomassie Blue stained. D to F, Corresponding membranes from each of the strains were immunoblotted with phospho-Thr antibodies. G, Corresponding membranes containing purified complexes from UWO 241 were probed with antibodies specific for various polypeptides, as indicated on the right. Molecular masses are indicated on the left (kilodaltons), and lane numbers are shown in brackets. Figure 4. Open in new tabDownload slide Immunoblot analysis of purified complexes from SAG 49.72, UWO 241, and 1690. A to C, Thylakoid membrane proteins and fractions isolated by Suc density centrifugation from SAG 49.72 (A), 1690 (B), and UWO 241 (C) were Coomassie Blue stained. D to F, Corresponding membranes from each of the strains were immunoblotted with phospho-Thr antibodies. G, Corresponding membranes containing purified complexes from UWO 241 were probed with antibodies specific for various polypeptides, as indicated on the right. Molecular masses are indicated on the left (kilodaltons), and lane numbers are shown in brackets. Band 4 exhibited a Chl a/b ratio between 4.98 and 8.05 in the two mesophilic strains, SAG 49.72 and 1690 (Table I), and absorption spectra consistent with a Chl a pigment-protein complex (Supplemental Fig. S2, A and B). This complex exhibited an unusual 77 K fluorescence emission spectrum, with a prominent emission maximum at 715 nm and an emission maximum of lower intensity between 680 and 683 nm in both SAG 49.72 and 1690 (Supplemental Fig. S2, E and F). In contrast, band 4 from UWO 241 exhibited a much lower Chl a/b ratio (2.58) than either SAG 49.72 or 1690 (Table I) and a major 77 K emission maximum at 680 nm with a shoulder at 712 nm (Fig. 3D). To confirm the tentative identification of the individual chlorophyll-protein complexes separated by Suc density centrifugation based on spectral analyses, we examined the polypeptide composition of each green band by SDS-PAGE for SAG 49.72 (Fig. 4A), 1690 (Fig. 4B), and UWO 241 (Fig. 4C) coupled with immunoblotting (Fig. 4G). Figure 4, A to C, indicates that band 1 consists of major polypeptides of about 24 kD that react positively with Lhcb3 antibodies (Fig. 4G). The spectral data combined with the polypeptide analyses and immunoblots are consistent with the identification of band 1 as the major LHCII of PSII in all three strains. SDS-PAGE of band 2 indicated that it contained three major polypeptides in the molecular mass range of 32 to 47 kD in all three strains (Fig. 4, A–C). The immunoblots indicated that the major polypeptides of the pigment-protein complex associated with band 2 are the 32-kD D1 (PsbA) and CP43 (Fig. 4G). The spectral data combined with the polypeptide analyses and immunoblots are consistent with the identification of band 2 as the PSII core-reaction center complex. However, band 2 also exhibited minor levels of the PSI subunits PsaA, PsaL, PsaC, and PsaD but no detectable levels of Lhcb3 (Fig. 4G). The major polypeptides associated with band 3 exhibited molecular masses in the 21-kD range, the 50- to 60-kD range, as well as a major, high-molecular-mass polypeptide greater than 100 kD (Fig. 4, A–C). The immunoblots confirmed that this chlorophyll-protein complex consisted of the major PSI reaction polypeptide, PsaA, the PSI subunits PsaL, PsaC, and PsaD, and Lhca2, but no PSII subunits (D1 or CP43) or Lhcb3 were detected in this fraction. The spectral data combined with the polypeptide analyses and immunoblots are consistent with the identification of band 3 as the PSI core-reaction center-LHCI complex. Similar to band 3, band 4 was a multisubunit Chl a pigment-protein complex in all three strains examined (Fig. 4, A–C). In the psychrophile UWO 241, band 4 contained the PSI-associated subunits (PsaA, PsaL, PsaC, and PsaD and Lhca2) but not the D1 or CP43 associated with PSII (Fig. 4G). Interestingly, band 4 also exhibited a relatively high abundance of the thylakoid protease FtsH (Fig. 4G). Since the 77 K emission spectrum of band 4 was distinct from that of band 3, we suggest that band 4 represents a PSI pigment-protein complex distinct from the PSI core-reaction center complex. Given that band 4 represents the highest density complex separated on the Suc gradient, we suggest that band 4 may represent a putative PSI supercomplex based on our spectral and polypeptide analyses. For all three algal strains tested, purified pigment-protein complexes were subjected to denaturation by SDS-PAGE (Fig. 4, A–C) followed by immunodetection with phospho-Thr antibodies to detect the phosphorylated subunits in each complex (Fig. 4, D–F). We detected no major phosphorylated polypeptides associated with the PSI core complexes from SAG 49.72 (Fig. 4D, band 3), 1690 (Fig. 4E, band 3), or UWO 241 (Fig. 4F, band 3). However, phosphorylation of Thr residues in SAG 49.72 (Fig. 4D) and C. reinhardtii (Fig. 4E) were detectable for the major Lhcb polypeptides of LHCII (Fig. 4, D and E, band 1) as well as the PSII core complex polypeptides D1 and CP43 (Fig. 4, D and E, band 2). Although the PSII core complex polypeptides D1 and CP43 (Supplemental Fig. S3, B and C) were phosphorylated in UWO 241 (Fig. 4F, band 2; Supplemental Fig. S3A), no phosphorylated LHCII polypeptides were detected in UWO 241, even in the purified LHCII complex (Fig. 4F, band 1). However, in contrast to either SAG 49.72 or 1690, UWO 241 exhibited two novel phosphorylated polypeptides (Fig. 4F, band 4) associated with the high-molecular-mass putative supercomplex: one high-molecular-mass band (greater than 130 kD) and one low-molecular-mass band of approximately 17 kD, which was also detected in the thylakoid fraction (Fig. 4F). BN-PAGE was repeated in order to compare the molecular mass and composition of the putative PSI supercomplex with the other Suc gradient purified complexes isolated from UWO 241 (Fig. 5A). LHCII, PSII, and the PSI+LHCI core complex exhibited the expected molecular masses of approximately 240 kD, approximately 450 kD, and approximately 700 kD, respectively (Fig. 5A, bands 1, 2, and 3, respectively). However, BN-PAGE confirmed that the putative PSI supercomplex was distinguishable from the PSI+LHCI complex (Fig. 5A, band 4). The PSI+LHCI complex remained largely intact as a single pigment-protein complex with a molecular mass of approximately 700 kD and only a small fraction present as an approximately 460-kD complex (Fig. 5A, band 3). In contrast, the putative PSI supercomplex (Fig. 5A, band 4) exhibited a major component of a molecular mass complex of approximately 460 kD, with the presence of two other smaller chlorophyll-protein subcomplexes of approximately 250 and 160 kD that were absent from the PSI+LHCI complex (Fig. 5A, band 3). This further supports our suggestion that this unique complex (Fig. 5A, band 4) is indeed a modified PSI complex. Figure 5. Open in new tabDownload slide BN-PAGE of thylakoid membrane complexes and second dimension analysis of the supercomplex fraction from UWO 241. A, UWO 241 complexes purified by Suc density centrifugation were separated on a 3% to 16% gradient native gel. B, Following separation of the supercomplex by BN-PAGE, the resulting gel strip was solubilized with SDS, and individual subunits were separated using SDS-PAGE and stained with Coomassie Blue. C to F, Corresponding membranes immunoblotted with various specific antibodies as indicated: PsaA (C), PsaD (D), phospho-Thr (E), and Cyt f (F). The locations of these proteins are represented by asterisks in B. Apparent molecular masses are indicated on the sides (kilodaltons). G, Absorbance difference spectrum (reduced minus oxidized) of Cyt f in the purified PSI supercomplex fraction of UWO 241. Figure 5. Open in new tabDownload slide BN-PAGE of thylakoid membrane complexes and second dimension analysis of the supercomplex fraction from UWO 241. A, UWO 241 complexes purified by Suc density centrifugation were separated on a 3% to 16% gradient native gel. B, Following separation of the supercomplex by BN-PAGE, the resulting gel strip was solubilized with SDS, and individual subunits were separated using SDS-PAGE and stained with Coomassie Blue. C to F, Corresponding membranes immunoblotted with various specific antibodies as indicated: PsaA (C), PsaD (D), phospho-Thr (E), and Cyt f (F). The locations of these proteins are represented by asterisks in B. Apparent molecular masses are indicated on the sides (kilodaltons). G, Absorbance difference spectrum (reduced minus oxidized) of Cyt f in the purified PSI supercomplex fraction of UWO 241. To examine the polypeptide composition of the two smaller chlorophyll-containing subcomplexes observed in Figure 5A (band 4), the purified putative PSI supercomplex was solubilized with SDS and separated in the second dimension by SDS-PAGE (Fig. 5B). Immunoblots confirmed the presence of PsaA (Fig. 5C) and PsaD (Fig. 5D) in the 460-kD pigment-protein complex and a 17-kD phosphorylated polypeptide (Fig. 5E). In addition, immunoblots of the polypeptides associated with the two smaller (approximately 250- and 160-kD) subcomplexes exhibited the presence of the Cyt f polypeptide (Fig. 5F). The 34-kD polypeptide identified as Cyt f by immunoblotting (Fig. 5F) matched the 34-kD polypeptide present on the Coomassie Blue gel (Fig. 5B, asterisk). A redox difference spectra of the purified PSI supercomplex indicated the presence of the Cyt b 6/f complex within this fraction (Fig. 5G). The presence of Cyt f within the supercomplex was also confirmed by heme staining, which indicated a heme structure associated with a 34-kD subunit as well as the large (greater than 130 kD) band (Supplemental Fig. S4). Consequently, the Cyt b 6/f complex appears to be a component of the putative PSI supercomplex of UWO 241. Stability of the Putative PSI Supercomplex To test the sensitivity of the putative PSI supercomplex to thylakoid protein phosphorylation status and its effects on the stability of pigment-protein complexes (Fig. 6), we examined the effects of the phosphatase inhibitor NaF and the protein kinase inhibitor staurosporine, a known inhibitor of protein kinases (Gangwani et al., 1996; Horling et al., 2001) and state transitions in C. reinhardtii (Morgan-Kiss et al., 2002b; Takahashi et al., 2006). UWO 241 pigment-protein complexes isolated in the presence of NaF resulted in the separation of four distinct bands including a high-density band of supercomplexes: LHCII, PSII core, PSI+LHCI, and the putative PSI supercomplex (Fig. 6A). In contrast, the supercomplex band was absent when thylakoid complexes were isolated in the presence of staurosporine, although the major chlorophyll-protein complexes associated with LHCII, PSII, and PSI+LHCI were still observed (Fig. 6A). Figure 6B confirms that staurosporine inhibits the phosphorylation of the specific polypeptides associated with the putative PSI supercomplex (polypeptides greater than 130 kD plus the 17-kD polypeptide) as well as the PSII subunits, CP43, and the 32-kD D1 polypeptide. These results indicate that the phosphorylation state of the thylakoid membrane proteins affect the stability of the putative PSI supercomplex of UWO 241. Figure 6. Open in new tabDownload slide Effects of inhibitors of thylakoid protein phosphorylation on the stability of the putative PSI supercomplex (SC) in UWO 241. A, Thylakoid membranes were either treated with the phosphatase inhibitor, NaF, or with the kinase inhibitor, staurosporine. B, Under these conditions, thylakoid membrane proteins separated by SDS-PAGE were probed with antibodies specific for phospho-Thr. C and D, Thylakoid membranes treated with either NaF (C) or staurosporine (D) were fractionated into 15 fractions of equal volume (0.8 mL), solubilized with SDS, and 20 μL of each fraction was separated by SDS-PAGE. E to H, Corresponding membranes were immunoblotted with antibodies specific for PsaA, PsaD, and Cyt f, as indicated. Figure 6. Open in new tabDownload slide Effects of inhibitors of thylakoid protein phosphorylation on the stability of the putative PSI supercomplex (SC) in UWO 241. A, Thylakoid membranes were either treated with the phosphatase inhibitor, NaF, or with the kinase inhibitor, staurosporine. B, Under these conditions, thylakoid membrane proteins separated by SDS-PAGE were probed with antibodies specific for phospho-Thr. C and D, Thylakoid membranes treated with either NaF (C) or staurosporine (D) were fractionated into 15 fractions of equal volume (0.8 mL), solubilized with SDS, and 20 μL of each fraction was separated by SDS-PAGE. E to H, Corresponding membranes were immunoblotted with antibodies specific for PsaA, PsaD, and Cyt f, as indicated. Fractionation of the Suc gradients containing thylakoids from UWO 241 treated with either NaF (Fig. 6C) or staurosporine (Fig. 6D) indicated that the phosphorylation state of the thylakoid membranes altered the migration patterns for both PsaA (Fig. 6, E and F) as well as Cyt f (Fig. 6, G and H). Consistent with the formation of the putative PSI supercomplex consisting of both PSI and Cyt b 6/f components in the presence of NaF, both PsaA and Cyt f were detected in the high-density fractions 4 to 7, coincident with the band for the putative PSI supercomplex on the Suc gradient (Fig. 6, E and G). However, the primary fractions containing the highest levels of PsaA and Cyt f were detected in the lower density fractions 7 to 10 and 10 to 12, respectively, in the presence of NaF. In contrast, in the presence of staurosporine, the pigment band associated with the putative PSI supercomplex (Fig. 6A) was not detectable, and minimal levels of PsaA and Cyt f were detected in the high-density fractions 4 to 7 (Fig. 6, F and H). Furthermore, PsaA and Cyt f were detected only in the lower density fractions 7 to 11 and 12 to 15, respectively (Fig. 6, F and H). UWO 241 is not only a psychrophile; it is also halotolerant (Takizawa et al., 2009). SDS-PAGE indicated that salt concentration had minimal effects on the thylakoid polypeptide complement of UWO 241 (Fig. 7A). However, phospho-Thr immunoblot analysis of total SDS-solubilized thylakoid membrane proteins indicated that growth at low salt (70 mm) resulted in a marked reduction in the phosphorylation status of both the 17-kD band and the large greater than 130-kD protein complex compared with cells grown in high salt (700 mm; Fig. 7B). Furthermore, Suc gradient purification of the thylakoid pigment-protein complexes from low-salt-grown cells resulted in an 86% reduction of the putative PSI supercomplex, based on chlorophyll content, compared with that of high-salt-grown cells (Fig. 7, C and D). Consistently, for low-salt-grown cells, the PSI subunit, PsaA, as well as Cyt f showed a strong reduction in abundance within fractions 4 and 5, containing the PSI supercomplex, compared with growth at high salt (Fig. 7E). Regardless of the salt concentration under which UWO 241 was grown, phosphorylation of the major LHCII was not detected, which is consistent with previous results (Takizawa et al., 2009). Figure 7. Open in new tabDownload slide Effects of growth salt concentration on phosphorylation. Total thylakoid membrane proteins of UWO 241 grown with high (700 mm) and low (70 mm) NaCl were loaded on an equal chlorophyll basis and separated by SDS-PAGE. A, Gel stained with Coomassie Blue. B, Corresponding membrane probed with antibodies specific for phospho-Thr. C and D, Subsequently, complexes of UWO 241 grown in either 70 mm (C) or 700 mm NaCl (D) were fractionated by Suc density centrifugation. E, Fractions 4 and 5, containing the supercomplex (SC), were probed with antibodies specific for Cyt f and PsaA under low- and high-salt treatments. Apparent molecular masses are indicated (kilodaltons). Figure 7. Open in new tabDownload slide Effects of growth salt concentration on phosphorylation. Total thylakoid membrane proteins of UWO 241 grown with high (700 mm) and low (70 mm) NaCl were loaded on an equal chlorophyll basis and separated by SDS-PAGE. A, Gel stained with Coomassie Blue. B, Corresponding membrane probed with antibodies specific for phospho-Thr. C and D, Subsequently, complexes of UWO 241 grown in either 70 mm (C) or 700 mm NaCl (D) were fractionated by Suc density centrifugation. E, Fractions 4 and 5, containing the supercomplex (SC), were probed with antibodies specific for Cyt f and PsaA under low- and high-salt treatments. Apparent molecular masses are indicated (kilodaltons). Identification of the Phosphoproteins of the Putative PSI Supercomplex The purified putative PSI supercomplex of UWO 241 was subjected to two-dimensional isoelectric focusing (IEF)-SDS-PAGE to isolate and purify the phosphorylated protein subunit(s) in preparation for sequencing by mass spectrometry (Fig. 8). IEF indicated the presence of at least four major phosphoproteins detected with phospho-Thr antibodies in the purified putative PSI supercomplex of UWO 241 (Fig. 8B, spots 1–4). Phosphoproteins 1 (pI = 8.5) and 2 (pI = 7.2) exhibited comparable molecular masses (17 kD) but different pI. Phosphoproteins 3 (70 kD) and 4 (greater than 130 kD) differed in molecular mass as well as pI (4.9 and 6.9, respectively). Figure 8. Open in new tabDownload slide Two-dimensional IEF/SDS-PAGE purification of phosphorylated polypeptides from the supercomplex of UWO 241. IEF (broad range, pI 3–10) was used to separate supercomplex subunits based on pI values in the first dimension. Resultant gel strips were equilibrated and separated on a 12% SDS-PAGE gel and stained with Coomassie Blue (A) or immunoblotted with phospho-Thr antibodies (B). Apparent molecular masses are indicated on the left (kilodaltons). Figure 8. Open in new tabDownload slide Two-dimensional IEF/SDS-PAGE purification of phosphorylated polypeptides from the supercomplex of UWO 241. IEF (broad range, pI 3–10) was used to separate supercomplex subunits based on pI values in the first dimension. Resultant gel strips were equilibrated and separated on a 12% SDS-PAGE gel and stained with Coomassie Blue (A) or immunoblotted with phospho-Thr antibodies (B). Apparent molecular masses are indicated on the left (kilodaltons). Phosphoproteins 1 to 4 were excised from the gel, digested, and analyzed by liquid chromatography isoelectric focusing nano-tandem mass spectrometry (nano-LC-ESI-MS/MS). For identification, the resultant peptide ions generated by nano-LC-ESI-MS/MS were searched against the National Center for Biotechnology Information databases using PEAKS (homology). Sequencing of the peptides originating from phosphoproteins 1 and 2 revealed that these two phosphoproteins were the same proteins in their peptide sequences that exhibited 94% identity. A database protein BLAST search of these peptide sequences revealed that these polypeptides belonged to the PsbP superfamily of proteins, and are hereafter referred to as PsbP-like proteins. Tyr-146 was identified as a phosphorylated residue on the PsbP-like protein from UWO 241 (Supplemental Fig. S5). Spots 1 and 2 showed significant sequence matches to a number of authentic PsbP proteins, which are summarized in Supplemental Table S1. Among these, PsbP (OXYGEN-EVOLVING ENHANCER PROTEIN2/OXYGEN-EVOLVING COMPLEX23) from C. reinhardtii showed the highest sequence similarity, with 70.6% of the amino acids being identical to the PsbP-like protein. Figure 9 shows the alignment of phosphoproteins 1 and 2 with amino acid sequences of authentic PsbP from C. reinhardtii, Arabidopsis, and Brassica rapa. Figure 9. Open in new tabDownload slide Alignment of the PsbP-like protein from the supercomplex of UWO 241 from spots 1 and 2 with the amino acid sequences of homologous authentic PsbP proteins from three other organisms. The alignment was performed using ClustalW. The GenBank accession numbers are as follows: C. reinhardtii, P11471.1; B. rapa, XP_009144275.1; and Arabidopsis, NP172153.1. Multiple phosphorylation sites (Ser-73, Ser-133, Ser-140, Ser-151, Thr-154, Ser-156, Ser-166, Ser-169, Ser-174, Thr-175, Thr-176, Thr-177, Ser-241, and Thr-243) that have been recently identified are indicated for PsbP1 (arrows) and PsbP4 (Ser-142; asterisk) in C. reinhardtii (Wang et al., 2014). The phosphorylated Tyr residue, Tyr-146, identified in UWO 241 during this study is indicated with the triangle. Figure 9. Open in new tabDownload slide Alignment of the PsbP-like protein from the supercomplex of UWO 241 from spots 1 and 2 with the amino acid sequences of homologous authentic PsbP proteins from three other organisms. The alignment was performed using ClustalW. The GenBank accession numbers are as follows: C. reinhardtii, P11471.1; B. rapa, XP_009144275.1; and Arabidopsis, NP172153.1. Multiple phosphorylation sites (Ser-73, Ser-133, Ser-140, Ser-151, Thr-154, Ser-156, Ser-166, Ser-169, Ser-174, Thr-175, Thr-176, Thr-177, Ser-241, and Thr-243) that have been recently identified are indicated for PsbP1 (arrows) and PsbP4 (Ser-142; asterisk) in C. reinhardtii (Wang et al., 2014). The phosphorylated Tyr residue, Tyr-146, identified in UWO 241 during this study is indicated with the triangle. The nano-LC-ESI-MS/MS analysis of the 70-kD phosphoprotein 3 revealed the presence of two polypeptides. Since these proteins had the same apparent molecular mass of 72 kD and pI of 4.9, they could not be separated by IEF. However, based on sequence analyses, the two polypeptides were identified as an ATP-dependent zinc metalloprotease FtsH from Medicago sativa (summarized in Supplemental Table S2) and a heat shock cognate 70-kD protein (HSP) as determined by the matching of five peptides from Arabidopsis (Supplemental Table S2). Although the PEAKS software had identified potential phosphorylation modification sites for all three proteins (PsbP-like, HSP, and FtsH), the ion scores of individual peptides were insufficient to confirm the specific phosphorylated Thr residues detected by immunoblotting. Only one phosphorylation was confirmed, Tyr-146 on the PsbP-like protein (spots 1 and 2) from the supercomplex of UWO 241 (Supplemental Fig. S5). Phosphoprotein 4 contained several different proteins, with 14 and nine peptide matches to PsaA and PsaB, respectively (summarized in Supplemental Table S3). Phosphoprotein 4 also showed the presence of an ATP-dependent FtsH metalloprotease, with three sequence matches, two of which were identical to those found in the 70-kD phosphoprotein 3. Therefore, the 70-kD phosphoprotein 3 is probably an FtsH metalloprotease, which was also present in the large (greater than 130 kD) protein complex and may be responsible for the positive phosphorylation signal associated with phosphoprotein 4. The presence of the individual polypeptides associated with the specific protein spots isolated by IEF (Fig. 8B) was confirmed by nano-LC-ESI-MS/MS analysis of the complete PSI supercomplex purified by Suc gradient centrifugation (Supplemental Table S4). In addition to the polypeptides associated with phosphoprotein spots 1 to 4 (Fig. 8B) in the complete, purified complex, we also detected an ADP-ribosylation factor associated with the ARF family of GTPases, RF4, a PSI assembly protein associated with the chlorophyll fluorescence mutant, Ycf4, superfamily in Chlamydomonas spp., an adenine nucleotide translocator, as well as the expected Cyt f from UWO 241 and the PsbP subunits. P700-Dependent Cyclic Electron Transport Far-red (FR) light-induced absorbance changes at 820 nm (ƊA 820-860) were used as an estimate of the extent of P700 photooxidation in vivo (Mi et al., 1992; Asada et al., 1993; Ivanov et al., 2000; Morgan-Kiss et al., 2002a). Illumination of the algal cells with FR light caused an increase in A 820-860 signal, which indicated the oxidation of P700 to the P700+ radical. Thus, steady-state FR light-induced absorbance changes (ƊA 820-860) were used to express the steady-state level of P700 photooxidation. Under control conditions, the mesophilic strain SAG 49.72 exhibited an almost 2-fold higher steady-state value of ƊA 820-860 than UWO 241 cells (Table II), which is in agreement with previous results (Morgan-Kiss et al., 2002a, 2002b). Effects of antimycin A (AA; 20 µm) on the FR light-induced steady-state oxidation of P700 and half-times for P700+ reduction after turning off the FR light in mesophilic (SAG 49.72) and psychrophilic (UWO 241) strains cultivated at different growth temperatures and either high-salt (HS; 700 mm) or low-salt (LS; 70 mm) concentration Table II. Effects of antimycin A (AA; 20 µm) on the FR light-induced steady-state oxidation of P700 and half-times for P700+ reduction after turning off the FR light in mesophilic (SAG 49.72) and psychrophilic (UWO 241) strains cultivated at different growth temperatures and either high-salt (HS; 700 mm) or low-salt (LS; 70 mm) concentration All data represent means ± se from four to 11 measurements in four to five independent experiments. All measurements were performed at the corresponding growth temperatures of 5°C (SAG 49.72) and 25°C (UWO 241) or at the same growth temperature of 16°C. Strain/Treatment . ƊA 820-860 . Half-Time for P700+ Reduction . . Control . +AA . Percentage of Control . Control . +AA . Percentage of Control . mV s SAG 49.72 (25°C) 14.2 ± 1.7 (8) 26.3 ± 1.6 (5) 185.1 3.4 ± 1.2 (7) 9.0 ± 0.4 (7) 266.7 SAG 49.72 (16°C) 15.7 ± 1.7 (9) 47.7 ± 8.1 (4) 303.8 10.1 ± 0.9 (9) 19.2 ± 3.5 (4) 190.1 UWO 241 (5°C; HS) 7.1 ± 1.4 (6) 21.9 ± 2.4 (10) 310.0 1.1 ± 0.2 (6) 8.2 ± 0.8 (10) 747.7 UWO 241 (16°C) 50.0 ± 7.6 (10) 82.2 ± 11.2 (5) 164.4 7.9 ± 0.8 (10) 9.3 ± 0.9 (5) 117.7 UWO 241 (5°C; LS) 29.9 ± 3.5 (11) 38.2 ± 3.4 (5) 127.7 2.7 ± 0.5 (11) 2.1 ± 0.1 (5) 77.8 Strain/Treatment . ƊA 820-860 . Half-Time for P700+ Reduction . . Control . +AA . Percentage of Control . Control . +AA . Percentage of Control . mV s SAG 49.72 (25°C) 14.2 ± 1.7 (8) 26.3 ± 1.6 (5) 185.1 3.4 ± 1.2 (7) 9.0 ± 0.4 (7) 266.7 SAG 49.72 (16°C) 15.7 ± 1.7 (9) 47.7 ± 8.1 (4) 303.8 10.1 ± 0.9 (9) 19.2 ± 3.5 (4) 190.1 UWO 241 (5°C; HS) 7.1 ± 1.4 (6) 21.9 ± 2.4 (10) 310.0 1.1 ± 0.2 (6) 8.2 ± 0.8 (10) 747.7 UWO 241 (16°C) 50.0 ± 7.6 (10) 82.2 ± 11.2 (5) 164.4 7.9 ± 0.8 (10) 9.3 ± 0.9 (5) 117.7 UWO 241 (5°C; LS) 29.9 ± 3.5 (11) 38.2 ± 3.4 (5) 127.7 2.7 ± 0.5 (11) 2.1 ± 0.1 (5) 77.8 Open in new tab Table II. Effects of antimycin A (AA; 20 µm) on the FR light-induced steady-state oxidation of P700 and half-times for P700+ reduction after turning off the FR light in mesophilic (SAG 49.72) and psychrophilic (UWO 241) strains cultivated at different growth temperatures and either high-salt (HS; 700 mm) or low-salt (LS; 70 mm) concentration All data represent means ± se from four to 11 measurements in four to five independent experiments. All measurements were performed at the corresponding growth temperatures of 5°C (SAG 49.72) and 25°C (UWO 241) or at the same growth temperature of 16°C. Strain/Treatment . ƊA 820-860 . Half-Time for P700+ Reduction . . Control . +AA . Percentage of Control . Control . +AA . Percentage of Control . mV s SAG 49.72 (25°C) 14.2 ± 1.7 (8) 26.3 ± 1.6 (5) 185.1 3.4 ± 1.2 (7) 9.0 ± 0.4 (7) 266.7 SAG 49.72 (16°C) 15.7 ± 1.7 (9) 47.7 ± 8.1 (4) 303.8 10.1 ± 0.9 (9) 19.2 ± 3.5 (4) 190.1 UWO 241 (5°C; HS) 7.1 ± 1.4 (6) 21.9 ± 2.4 (10) 310.0 1.1 ± 0.2 (6) 8.2 ± 0.8 (10) 747.7 UWO 241 (16°C) 50.0 ± 7.6 (10) 82.2 ± 11.2 (5) 164.4 7.9 ± 0.8 (10) 9.3 ± 0.9 (5) 117.7 UWO 241 (5°C; LS) 29.9 ± 3.5 (11) 38.2 ± 3.4 (5) 127.7 2.7 ± 0.5 (11) 2.1 ± 0.1 (5) 77.8 Strain/Treatment . ƊA 820-860 . Half-Time for P700+ Reduction . . Control . +AA . Percentage of Control . Control . +AA . Percentage of Control . mV s SAG 49.72 (25°C) 14.2 ± 1.7 (8) 26.3 ± 1.6 (5) 185.1 3.4 ± 1.2 (7) 9.0 ± 0.4 (7) 266.7 SAG 49.72 (16°C) 15.7 ± 1.7 (9) 47.7 ± 8.1 (4) 303.8 10.1 ± 0.9 (9) 19.2 ± 3.5 (4) 190.1 UWO 241 (5°C; HS) 7.1 ± 1.4 (6) 21.9 ± 2.4 (10) 310.0 1.1 ± 0.2 (6) 8.2 ± 0.8 (10) 747.7 UWO 241 (16°C) 50.0 ± 7.6 (10) 82.2 ± 11.2 (5) 164.4 7.9 ± 0.8 (10) 9.3 ± 0.9 (5) 117.7 UWO 241 (5°C; LS) 29.9 ± 3.5 (11) 38.2 ± 3.4 (5) 127.7 2.7 ± 0.5 (11) 2.1 ± 0.1 (5) 77.8 Open in new tab Since, under FR illumination, PSI is the primary photosystem that is operational, the kinetics of P700+ reduction when the FR light is turned off is presumed to reflect primarily the rates of CEF around PSI (Maxwell and Biggins, 1976, Ravenel et al., 1994) and/or the interaction of stromal components with the intersystem electron transport chain (Asada et al., 1992). The rates of P700+ reduction in the dark were 3-fold faster in UWO 241 (1.1 s) compared with SAG 49.72 (3.4 s; Table II), indicating an increased capacity for CEF around PSI and/or increased interaction between stromal electron donors and intersystem electron transport in UWO 241 compared with the mesophilic SAG 49.72 strain. To confirm this, AA was used as an effective inhibitor of the AA-sensitive ferredoxin-plastoquinone reductase (FQR)-dependent pathway for CEF (Moss and Bendall, 1984; Shikanai, 2007; Antal et al., 2013). Although blocking the FQR-dependent CEF pathway with AA resulted in a comparable level of ƊA 820-860, the presence of AA inhibited the rates of P700+ reduction in the dark 7.5-fold in UWO 241 but only 2.7-fold in SAG 49.72 (Table II), thus confirming the significantly greater stimulation of CEF in UWO 241 relative to SAG 49.72 cells. Since, under control conditions, the steady-state level of P700+ UWO 241 was twice lower compared with SAG 49.72, preventing the influx of electrons to the intersystem electron transport chain by blocking the CEF with AA caused a differential increase in P700 photooxidation in UWO 241 (3-fold) versus SAG 49.72 (2-fold) such that ƊA 820-860 was comparable in UWO 241 and SAG 49.72 at their optimal growth temperatures of 25°C and 5°C (Table II). While the oxidation of P700 under FR light almost exclusively reflects the excitation of PSI, the extent of P700 photooxidation under white actinic light (AL) is affected by the excitation of both PSII and PSI (Asada et al., 1993). The contribution of linear electron transport from PSII to P700+ was estimated by measuring the extent of steady-state P700 photooxidation induced by white AL in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; Table III). Under white AL excitation in the absence of DCMU (P700+ control), electrons derived from PSII continuously reduced P700+ and generated a ƊA 820-860 that was 18% greater in UWO 241 than in SAG 49.75 (Table III). Preventing the electron flow from PSII to P700+ in the presence of the PSII inhibitor DCMU caused a 47% increase of the steady-state level of P700+ (Table III) in SAG 49.72, suggesting that electrons available for P700+ reduction originated from PSII and were delivered to P700+ via intersystem electron transport. In contrast, UWO 241 cells exhibited only 12% increase of P700+ in the presence of DCMU (Table III). This indicates that a greater fraction of electrons available for P700+ reduction must have been derived from sources other than PSII and that the contribution of PSII and intersystem electron transport to the reduction of P700+ was limited in the psychrophile UWO 241 compared with the mesophile SAG 49.72. Effects of DCMU on the level of P700+ under white AL (150 mmol photons m−2 s−1) excitation in the mesophilic (SAG 49.72) and psychrophilic (UWO 241) strains Table III. Effects of DCMU on the level of P700+ under white AL (150 mmol photons m−2 s−1) excitation in the mesophilic (SAG 49.72) and psychrophilic (UWO 241) strains The extent of P700 photooxidation was estimated by the AL-induced ƊA 820-860 in the absence and presence of 40 μm DCMU to inhibit the electron flow from PSII. All values represent means ± se from nine to 13 measurements in three independent experiments. All measurements were performed at the corresponding growth temperatures for UWO 241 (5°C) and SAG 49.72 (25°C). Strain . ƊA 820-860 . Absorbance Change Ratio, DCMU to Control . . Control . DCMU . . mV SAG 49.72 84.09 ± 3.89 (9) 124.16 ± 3.08 (13) 1.47 UWO 241 99.75 ± 2.66 (11) 111.75 ± 4.35 (11) 1.12 Strain . ƊA 820-860 . Absorbance Change Ratio, DCMU to Control . . Control . DCMU . . mV SAG 49.72 84.09 ± 3.89 (9) 124.16 ± 3.08 (13) 1.47 UWO 241 99.75 ± 2.66 (11) 111.75 ± 4.35 (11) 1.12 Open in new tab Table III. Effects of DCMU on the level of P700+ under white AL (150 mmol photons m−2 s−1) excitation in the mesophilic (SAG 49.72) and psychrophilic (UWO 241) strains The extent of P700 photooxidation was estimated by the AL-induced ƊA 820-860 in the absence and presence of 40 μm DCMU to inhibit the electron flow from PSII. All values represent means ± se from nine to 13 measurements in three independent experiments. All measurements were performed at the corresponding growth temperatures for UWO 241 (5°C) and SAG 49.72 (25°C). Strain . ƊA 820-860 . Absorbance Change Ratio, DCMU to Control . . Control . DCMU . . mV SAG 49.72 84.09 ± 3.89 (9) 124.16 ± 3.08 (13) 1.47 UWO 241 99.75 ± 2.66 (11) 111.75 ± 4.35 (11) 1.12 Strain . ƊA 820-860 . Absorbance Change Ratio, DCMU to Control . . Control . DCMU . . mV SAG 49.72 84.09 ± 3.89 (9) 124.16 ± 3.08 (13) 1.47 UWO 241 99.75 ± 2.66 (11) 111.75 ± 4.35 (11) 1.12 Open in new tab Since the stability of the PSI supercomplex of UWO 241 was greater when cells were grown under high salt rather than low salt (Fig. 7), we examined the effect of salt concentration on the P700 photooxidation signal (ƊA 820-860) and the dark relaxation kinetics of P700+ in UWO 241. The results in Table II show that, in UWO 241 grown at 5°C and high salt, the rates of P700+ reduction in the dark were 2.4-fold faster (1.1 s) than for UWO 241 grown at 5°C but low salt (2.7 s). Thus, the growth of UWO 241 at low salt inhibited the rate of PSI CEF. However, treatment of UWO 241 cells with the kinase inhibitor staurosporine did not result in significant reductions in rates of P700+ reduction in the dark (data not shown). DISCUSSION Photosynthetic organisms typically display a characteristic phosphorylation profile of specific thylakoid membrane polypeptides, the majority of which are associated with PSII (Owens and Ohad, 1982; Zer and Ohad, 2003). Phosphorylated polypeptides associated with the PSII core reaction center complex that undergo phosphorylation include the D1/D2 reaction center polypeptides as well as a 43-kD Chl a-binding protein (CP43; Hansson and Vener, 2003; Turkina et al., 2006; Vener, 2006, 2007; Yokthongwattana and Melis, 2006; Edelman and Mattoo, 2008). The phosphorylation of D1/D2 and CP43 is associated with the disassembly, degradation, and resynthesis of the D1 protein during the PSII repair cycle in response to PSII chronic photoinhibition (Yokthongwattana and Melis, 2006; Edelman and Mattoo, 2008; Tikkanen et al., 2008; Fristedt et al., 2009). Several proteins of the PSII light-harvesting antenna are also important sites of thylakoid protein phosphorylation that regulate energy distribution between PSII and PSI through state transitions in plants and green algae (Rochaix, 2011, 2014) and also serve to protect PSI under fluctuating irradiance (Grieco et al., 2012). These polypeptides include the LHCII complex components, Lhcbm1 and Lhcbm10, and a minor LHCII antennae subunit, CP29 (Lhcb4), during state transitions in C. reinhardtii (Kargul et al., 2005; Turkina et al., 2006; Vener, 2007) and Lhcb1, Lhcb2, and Lhcb4 in terrestrial plants (Bergantino et al., 1995, 1998; Tikkanen et al., 2006). In addition, Lhcbm4, Lhcbm6, Lhcbm9, Lhcbm11, and CP26 (Lhcb5) have also been shown to undergo phosphorylation during high-light exposure in C. reinhardtii (Turkina et al., 2006; Vener, 2007). However, in addition to the regulation of state transitions, thylakoid protein phosphorylation has been shown to be crucial for the acclimation to high light and anoxia in C. reinhardtii (Bergner et al., 2015) as well as to low light and fluctuating irradiance in Arabidopsis (Piippo et al., 2006; Tikkanen et al., 2006; Grieco et al., 2012). Although the phosphorylation of PSI polypeptides appears to be quite rare (Vener, 2006, 2007), PsaD, an extrinsic stroma-exposed protein, was reported as the first phosphorylated PSI subunit in Arabidopsis (Hansson and Vener, 2003). Recently, an alternative interpretation for the role of PSII-LHCII protein phosphorylation has been suggested based, in part, on several reports that indicate that LHCII, rather than being associated primarily with PSII, serves as an antenna pigment bed for both PSII and PSI during the acclimation of Arabidopsis to irradiance (Kouřil et al., 2013; Wientjes et al., 2013; Grieco et al., 2015). Furthermore, it appears that changes in light intensity induce alterations in the level of thylakoid protein phosphorylation independently of state transitions (Tikkanen and Aro, 2012; Mekala et al., 2015). Thus, a new model has emerged whereby PSII and PSI are embedded in a common lake of LCHII and, consequently, are energetically coupled to one another. Consequently, excess energy supplied to PSII-LHCII may be quenched by PSI through energy spillover (Papageorgiou and Govindjee, 2011; Tikkanen and Aro, 2014). STN8 appears to be the primary thylakoid protein kinase that regulates the phosphorylation of the PSII core proteins, whereas STN7 is involved in the phosphorylation of Lhcb1, Lchb2, and Lhcb4 in Arabidopsis (Bonardi et al., 2005; Tikkanen et al., 2008; Fristedt et al., 2009; Rochaix, 2011, 2014). The orthologous thylakoid protein kinases in C. reinhardtii are STT8 and STT7 (Depège et al., 2003; Vener, 2006; Rochaix, 2011, 2014). The activities of these thylakoid protein kinases are light dependent and regulated via the redox state of the intersystem photosynthetic electron transport chain (Bonardi et al., 2005; Tikkanen et al., 2006, 2008, 2012; Vener, 2006; Rochaix, 2011). Consistent with other mesophilic photosynthetic organisms such as 1690 and SAG 49.72, UWO 241 exhibits phosphorylation of the two PSII core proteins, CP43 and D1. This is in agreement with previous studies that show that UWO 241 exhibits rapid rates of D1 repair and recovery from photoinhibition at low temperatures (Pocock et al., 2007). Consequently, it appears that STT8 is present and active in UWO 241. However, in contrast to other photosynthetic organisms, UWO 241 does not display phosphorylation of PSII-associated LHCII polypeptides, which is consistent with our previous reports that UWO 241 is a natural green algal variant that does not exhibit the capacity to undergo state transitions and appears to be locked in state I (Morgan-Kiss et al., 2001b; Gudynaite-Savitch et al., 2006, 2007; Szyszka et al., 2007; Takizawa et al., 2009). We report here, to our knowledge for the first time, that this Antarctic psychrophile preferentially phosphorylates a pigment-protein supercomplex with a molecular mass of about 1,000 kD rather than LHCII components. Absorption spectroscopy, 77 K fluorescence emission spectroscopy, and immunoblot and nano-LC-ESI-MS/MS analyses are consistent with the identification of this pigment-protein complex as a PSI supercomplex. The PSI supercomplex from UWO 241 consists of components of both PSI and the Cyt b 6/f complex. We were able to detect and identify four specific phosphorylated polypeptides associated with this PSI supercomplex. Two phosphorylated polypeptides with a comparable molecular mass of 17 kD but different pI values were identified by nano-LC-ESI-MS/MS to be PsbP-like proteins. The third phosphorylated polypeptide was identified as a putative ATP-dependent zinc metalloprotease, FtsH. In Arabidopsis, thylakoid FtsH proteases were found to form a heterooligomeric hexameric complex and are believed to play an important role in protein degradation and development. The most studied function of the thylakoid FtsH protease complex is the degradation of the PSII reaction center protein, D1 (Kato et al., 2009); however, other substrates have been reported, such as the degradation of the Rieske-FeS subunit in pea (Pisum sativum) and the regulation of cytochrome b 6 levels in Chlamydomonas spp. There is also evidence that FtsH acts as a molecular chaperone involved in protein assembly. Furthermore, Stael et al. (2012) demonstrated that FtsH proteases, VARIEGATED1 (VAR1) and VAR2, are phosphorylated in a calcium-dependent manner in pea and Arabidopsis. Interestingly, PsbP, PsbQ1 and PsbQ2, and the calcium-sensing protein CAS and several PSI subunits (PsaN, PsaH, and PsaP) were also shown to exhibit calcium-dependent phosphorylation (Stael et al., 2012). The phosphorylation state of thylakoid membranes is the net effect of the combined rates of protein phosphorylation by thylakoid kinases such as the STT7/STT8 kinases versus the rates of dephosphorylation by thylakoid protein phosphatases such as PPH1/TAP38 (Rochaix, 2011). The stability of the PSI supercomplex in UWO 241 appears to be sensitive to the phosphorylation state of the phosphoprotein subunits, since the abundance of this complex is greatest when purified in the presence of NaF, which maximizes thylakoid polypeptide phosphorylation by inhibiting thylakoid phosphatases. Conversely, the abundance of the PSI supercomplex is minimal when it is purified in the presence of the kinase inhibitor staurosporine (Fig. 6). Thus, phosphorylation of the PSI supercomplex in UWO 241 may potentially play a role in providing structural stability to this pigment-protein complex. This is consistent with our previous report, which indicated that the phosphorylation status of the high-molecular-mass polypeptide complex of UWO 241 thylakoids is light dependent (Szyszka et al., 2007). In addition to polypeptide phosphorylation status, the maximum stability of the PSI supercomplex was also dependent upon the salt concentration under which UWO 241 cells were grown: high salt stabilized whereas low salt destabilized the PSI supercomplex. The latter is consistent with the fact that UWO 241 is not only a psychrophile but is also halotolerant and adapted to the high salt concentrations present in Lake Bonney, Antarctica, from which it was isolated (Morgan-Kiss et al., 2006; Takizawa et al., 2009). Several recent reports involving thylakoid membrane complex isolation have revealed existence of PSI supercomplexes. Peng et al. (2008) reported a novel NADPH DEHYDROGENASE (NDH)-PSI supercomplex with a molecular mass greater than 1,000 kD in Arabidopsis, which was composed almost exclusively of the PSI subunits as well as NDH subunits. Takahashi et al. (2006) used Suc density centrifugation to isolate PSI-LHCI supercomplexes from C. reinhardtii cells locked in either state I or state II and observed the reorganization of several LHCII subunits among the two photosystems during state transitions. Lemeille et al. (2009) examined the role of the STT7 kinase during state transitions in C. reinhardtii and reported that STT7 was associated with a large-M r complex that cofractionated with subunits of both the Cyt b 6/f complex and PSI but not with subunits of PSII. Iwai et al. (2010) identified a supercomplex in C. reinhardtii that governs CEF. This complex is composed of PSI-LHCI, LHCII, the Cyt b 6/f complex, FERREDOXIN-NADPH OXIDOREDUCTASE (FNR), and the PGRL1 protein and was isolated as a high-density band following Suc density gradient centrifugation of thylakoid membranes treated with n-tridecyl-β-d-maltoside (Iwai et al., 2010). Terashima et al. (2012) suggested that CEF is calcium dependent and regulated by three proteins: ANAEROBIC RESPONSE1, CAS, and PGRL1, which are part of a multiprotein, PSI-Cyt b 6/f complex in C. reinhardtii. Consistent with our study, the authors found that PsbP, PsbQ, as well as FtsH1 and FtsH2 were also associated with their CEF supercomplex (Terashima et al., 2012). However, the PSI supercomplex of UWO 241 is phosphorylated, and we identified the three specific phosphorylated subunits of the PSI supercomplex to be an ATP-dependent zinc metalloprotease FtsH and two PsbP-like polypeptides (Fig. 9; Supplemental Tables S1 and S2). We are unaware of any previous reports indicating that PSI supercomplexes are regulated by protein phosphorylation. A unique feature of the polypeptide composition of the PSI supercomplex in UWO 241 is the presence of two PsbP-like proteins that are reversibly phosphorylated. However, Yang et al. (2003) reported that OXYGEN-EVOLVING ENHANCER PROTEIN2 is phosphorylated via the cytoplasmic kinase domain of WAK1, a member of the WAK family of cell wall-associated receptor kinases in Arabidopsis. Authentic PsbP and PsbQ proteins typically are lumenal PSII subunits of the OXYGEN-EVOLVING COMPLEX (OEC) and play a role in stabilizing the manganese cluster (Ghanotakis et al., 1984; Miyao and Murata, 1985; Ifuku et al., 2008). PsbP (OEC23) has also been shown to be required for PSII core assembly, stability, and function (Ifuku et al., 2005; Yi et al., 2007). In addition to the authentic PsbP genes, recent genomic and proteomic studies have demonstrated the existence of many PsbPs in the chloroplast of higher plants (Ishihara et al., 2008; Ifuku et al., 2010). In Arabidopsis, there are eight PsbP homologs, all of which contain a thylakoid lumen-targeting signal (Ishihara et al., 2007, 2008; Ifuku et al., 2008, 2010). Based on similarity to the PsbP1 sequence (near 40%), two PsbP homologs have been referred to as PsbP-like (PPL) proteins, and the other PsbP homologs have been named PsbP domain (PPD) proteins (Ishihara et al., 2007, 2008). Alignment of amino acid sequences with that of the cyanobacterial PsbP homolog cyanoP revealed that both PPL homologs and PPD1 show significant homology to cyanoP (greater than 40% similarity), indicating that they are most likely of cyanobacterial origin (Ishihara et al., 2008). However, recently, PPD1 was shown to be essential for PSI assembly in Arabidopsis (Liu et al., 2012), a function unrelated to that of authentic PsbP proteins, which are components of PSII. Yeast two-hybrid assays revealed that PPD1 interacts specifically with PsaA and PsaB of PSI and acts at the posttranslational level (Liu et al., 2012). Furthermore, an Arabidopsis mutant of PPD5 exhibited decreased levels of NDH activity (Roose et al., 2011). The function of PPD6 is not unknown, but it is unique compared with other PsbP homologs, as it contains an intramolecular disulfide bond that may indicate sensitivity to redox regulation (Hall et al., 2012). The molecular functions of two PsbP-like homologs, PPL1 and PPL2, were also recently examined and compared with that of PsbP (Ishihara et al., 2007). Although both PPL proteins were involved in maintaining primary electron flow during plant stress responses, their functions were found to be diverse (Ishihara et al., 2007). Unexpectedly, PPL2 was shown to be the first subunit required for the accumulation of the chloroplast NDH complex in the thylakoid lumen (Ishihara et al., 2007). In addition, the function of either PPL1 or PPL2 did not overlap with that of authentic PsbPs, and neither protein was tightly associated with PSII (Ishihara et al., 2007). More recent studies have also confirmed that PPL2 (17 kD) is a novel subunit associated with the NDH complex in Arabidopsis (Peng et al., 2009; Suorsa et al., 2009). In addition, several PsbQ-like (PQL) proteins (including PsbQ-F1 and PsbQ-F2, both 17 kD) have been shown to be necessary for the function of the NDH complex in Arabidopsis (Majeran et al., 2008; Peng et al., 2009; Suorsa et al., 2009; Yabuta et al., 2010). Furthermore, PQL1 and PQL2 were found to be tightly associated with the NDH-PSI supercomplex (Suorsa et al., 2010; Yabuta et al., 2010). Thus, it appears that the majority of PPL and PQL polypeptides are required for the accumulation, stabilization, and activity of the NDH complex (Ishihara et al., 2008; Yabuta et al., 2010) as well as PSI (Liu et al., 2012). A comparison of the mass spectrometry-derived PsbP-like peptide sequence from UWO 241 shows 17% identity to PPL1/PPL2 in Arabidopsis and 14% to 19% identity to PsbP5 and PsbP3 in C. reinhardtii (Supplemental Fig. S6). In contrast to authentic PsbP subunits, PPLs and PQLs are not associated with PSII. Thus, we suggest that the identified PsbP-like phosphoproteins of UWO 241 may share a similar function in stabilizing the PSI supercomplex of UWO 241. However, our nano-LC-ESI-MS/MS analyses did not detect the presence of an NDH complex associated with the PSI supercomplex of UWO 241. Linear electron flow produces NADPH plus ATP, whereas CEF recycles photosynthetic electrons around PSI to generate ATP only (Finazzi et al., 1999; Eberhard et al., 2008; Cardol et al., 2011). Two CEF pathways have been proposed: (1) the NDH-dependent pathway and (2) an FQR pathway (Shikanai, 2007; Eberhard et al., 2008; Cardol et al., 2011; Johnson, 2011). The NDH pathway appears to be important in plants, where a chloroplastic, type I Ndh complex (NDH) similar to that found in plant mitochondria participates in CEF in combination with the Cyt b 6/f complex. Similarly, the cyanobacterium Synechocystis sp. PCC 6803 exhibits four distinct NDH1 complexes that govern PSI-mediated CEF (Battchikova et al., 2011; Bernát et al., 2011). However, the chloroplast genome of green algae lacks the genes that encode the chloroplastic Ndh complex (Maul et al., 2002; Oudot-LeSecq et al., 2007); thus, it has been suggested that, in green algae, CEF does not proceed through such a complex (Peltier and Cournac, 2002). However, nonphotochemical reduction of the plastoquinone pool is observed in green algae (Mus et al., 2005), which may be regulated by a type II NAD(P)H dehydrogenase (Nda2) in C. reinhardtii (Jans et al., 2008; Cardol et al., 2011). The FQR pathway, unlike the NDH pathway, is sensitive to AA, but the precise nature of FQR remains unknown. However, it has been suggested that PGR5 and PGRL1, first identified in Arabidopsis (Shikanai, 2007) and present in the C. reinhardtii genome (Merchant et al., 2007), may encode regulatory components of CEF in green algae (Cardol et al., 2011). It has also been proposed that the PGRL1/PGR5 complex, together with ferredoxin and FNR, facilitate AA-sensitive CEF in vascular plants (DalCorso et al., 2008). Recently, Sugimoto et al. (2013) suggested that the site of AA inhibition is PGR5 or proteins in close proximity to PGR5. Consistently, inactivation of a cyanobacterial gene that displays homology to PGR5 appears to disrupt AA-sensitive CEF (Yeremenko et al., 2005). However, although homologs of both PGRL1 and PGR5 exist in C. reinhardtii, it has been suggested that Chlamydomonas spp. CEF does not appear to be sensitive to AA (Iwai et al., 2010). In contrast, it appears that AA is effective in the inhibition of PSI CEF in the Antarctic psychrophile UWO 241 (Table II). Based on spectroscopic assessments (Fig. 3; Supplemental Fig. S2), polypeptide composition (Fig. 2), and immunoblots (Fig. 4), BN-PAGE (Fig. 5), and nano-LC-ESI-MS/MS of the high-density pigment-protein complex purified by Suc density centrifugation, we conclude that the Antarctic psychrophile exhibits a novel PSI-Cyt b 6/f supercomplex that represents the primary site of thylakoid protein phosphorylation and whose stability is dependent upon the thylakoid protein phosphorylation status as well as salt concentration. What is the functional role of the PSI supercomplex in UWO 241? Based on the P700 measurements (Tables II and III), we conclude that the presence of the novel PSI-Cyt b 6/f supercomplex contributes to enhanced CEF in UWO 241. We suggest that the presence of this PSI supercomplex in UWO 241 coupled with high rates of CEF reflects an adaptation to low-temperature and high-salt conditions that compensates for the fact that this psychrophile is locked in state I and lacks the ability to undergo state transitions. Alternatively, our data indicate that the organization of PSI and PSII within the thylakoid membrane is quite distinct in UWO 241, as indicated by the absence of the PSI 77 K fluorescence emission band at 712 nm in either isolated cells (Morgan-Kiss et al., 2002b) or purified thylakoid membranes (Fig. 3C). However, the PSI emission band at 712 nm is clearly evident in purified PSI (Fig. 3D). Thus, it appears that the Chl a fluorescence emission normally associated with PSI is quenched in intact cells and thylakoid membranes, which may be due to a distinct organization of PSI and PSII in UWO 241. Thus, the distinct thylakoid protein phosphorylation pattern and the apparent quenched state of PSI fluorescence emission may reflect a reorganization of the photosynthetic apparatus whereby excess excitation of PSII-LHCII is dissipated via energy spillover based on the recent model of Tikkanen and Aro (2014) rather than by state transitions. In support of this alternative, the strong nonphotochemical quenching capacity and the potential role for energy spillover in intact cells of UWO 241 have been noted previously (Szyszka et al., 2007; Takizawa et al., 2009). In conclusion, we suggest that the reversible phosphorylation of the PSI-Cyt b 6/f supercomplex in the Antarctic psychrophile Chlamydomonas sp. UWO 241 plays a role in the dynamic modulation between linear and cyclic electron transport, which is dependent upon the phosphorylation status of the specific phosphoprotein subunits (two PsbP-like polypeptides and an ATP-dependent zinc metalloprotease FtsH) of the PSI supercomplex as well as the presence of high salt concentrations. The CEF pathway in the psychrophile may be used to balance the excitation energy of the two photosystems and provide a constant supply of ATP, just as state transitions balance excitation energy during linear electron flow. Therefore, due to adaptation to its unique Antarctic environment of low temperature combined with low irradiance but high salt, we suggest that Chlamydomonas sp. UWO 241 favors the regulation of energy balance via CEF through the phosphorylation of a PSI supercomplex and/or energy spillover rather than via the regulation of state transitions through the phosphorylation of PSII-associated LHCII proteins. MATERIALS AND METHODS Growth Conditions Chlamydomonas raudensis SAG 49.72 and Chlamydomonas reinhardtii 1690 were grown axenically in Bold’s basal medium (BBM) at 24°C, whereas the psychrophilic strain Chlamydomonas sp. UWO 241 was grown in BBM supplemented with 0.7 m NaCl at 5°C. All cell cultures were aerated continuously under ambient CO2 conditions in 4-L glass Pyrex bottles and grown under an irradiance of 250 μmol photons m−2 s−1, which was generated by fluorescent tubes (Sylvania CW-40). To assess the effects of salt concentration on the structure and organization of PSI, UWO 241 cells were grown in BBM medium supplemented with either 70 or 700 mm NaCl. Midlog phase cells were used in all experiments. BN-PAGE Cells were disrupted by passing the suspension twice through a chilled French press at 6,000 p.s.i. Thylakoid membranes were purified through a Suc step gradient centrifugation procedure as described previously (Chua and Bennoun, 1975). Isolated thylakoid membranes were diluted in deionized water at a chlorophyll concentration of 2 mg mL−1 and solubilized with 2% (w/v) DDM (Sigma) on ice for 30 min and centrifuged at 20,000g for 30 min to remove unsolubilized material. The supernatant was supplemented with 0.25 volume of sample buffer (50 mm BisTris-HCl, pH 7.2, 50 mm NaCl, 10% [w/v] glycerol, and 0.001% [w/v] Ponceau S) and 0.1 volume of a solution containing 5% (w/v) Serva Blue G and 750 mm ε-aminocaproic acid and subjected to BN-PAGE using 4% to 16% (w/v) acrylamide gradient gels (Invitrogen). Electrophoresis was performed at 4°C at 15 mA for 1.5 h. For protein separation in the second dimension, the lanes were excised and incubated with 1% (v/v) β-mercaptoethanol and 1% (w/v) SDS for 10 min at room temperature, rinsed with deionized water, and subjected to SDS-PAGE as described below. NaF was present at a final concentration of 20 mm throughout the thylakoid isolation and purification procedure, except where noted, to inhibit any phosphatase activity and to ensure maximum protein phosphorylation. SDS-PAGE and Immunoblotting Thylakoids were isolated as described (Morgan-Kiss et al., 2005). Total thylakoid preparations from all three Chlamydomonas spp. strains were solubilized in a 60 mm Tris (pH 7.8) buffer containing 1 mm EDTA, 12% (w/v) Suc, 1% (w/v) dithiothreitol (DTT), and 2% (w/v) SDS to attain an SDS:chlorophyll ratio of 20:1. Total thylakoid samples were loaded on an equal chlorophyll basis. Purified complex fractions isolated by Suc density centrifugation were solubilized with 1% (w/v) SDS and 1% (v/v) β-mercaptoethanol and loaded on a chlorophyll basis with the following content: thylakoids, 1.4 μg; LHCII, 0.6 μg; PSII, 0.5 μg; PSI, 1.1 μg; and supercomplex, 0.9 μg (per lane). These concentrations were used to achieve optimal detection of complex subunits and were calculated based on chlorophyll-protein ratios and estimated protein number/complex values. Electrophoresis was performed using a Mini-Protean II apparatus (Bio-Rad) with a 12% (w/v) polyacrylamide resolving gel, containing 6 m urea and 0.66 m Tris (pH 8.8), and an 8% (w/v) polyacrylamide stacking gel, containing 0.125 m Tris (pH 6.8) using the buffer system of Laemmli (1970). In-gel heme staining was detected by peroxidase activity in the presence of 3,3′,5,5′-tetramethylbenzidine as described previously (Thomas et al., 1976). Proteins separated by SDS-PAGE were stained with Coomassie Blue or transferred electrophoretically to nitrocellulose membranes (Bio-Rad; 0.2-μm pore size) at 100 V for 1 h at 5°C. The membranes were preblocked with a Tris-buffered salt (20 mm Tris, pH 7.5, and 150 mm NaCl) containing 5% (w/v) skim milk powder and 0.01% (v/v) Tween 20. Membranes were probed with various antibodies as indicated in the figures at the following dilutions: PsaA at 1:2,000; PsaD at 1:5,000; PsaC at 1:2,000; Lhca2 at 1:2,000; PsaL at 1:5,000; D1 at 1:5,000; Cyt f at 1:2,000; Lhcb3 at 1:2,000; and CP43 at 1:2,000. Polyclonal phospho-Thr antibody (Zymed Laboratories) was used at 1:500 dilution to immunodetect thylakoid phosphoproteins. After incubation with secondary antibodies conjugated with horseradish peroxidase (Sigma), the antibody-protein complexes were visualized using enhanced chemiluminescence detection reagents (GE Healthcare). Suc Density Gradient Centrifugation Cells were disrupted using a French press as described above. Thylakoid membranes were purified through a Suc step gradient centrifugation procedure as described (Chua and Bennoun, 1975). Purified thylakoid membranes were resuspended in deionized water at a chlorophyll concentration of 0.9 mg mL−1, solubilized with 1% (w/v) DDM on ice for 25 min, and centrifuged to remove unsolubilized material. Purified thylakoid membranes were loaded on a continuous 1.3 to 0.1 m Suc density gradient and ultracentrifuged as described previously (Takahashi et al., 1991). All buffers for both thylakoid isolation and complex purification were supplemented with either 20 mm NaF or 0.2 μm staurosporine. Separated bands containing each complex were fractionated. Complexes were diluted three times with 20 mm HEPES, pH 7.5, and pelleted by centrifugation at 150,000g for 8 h. IEF Concentrated fractions of the PSI supercomplex were precipitated with 4 volumes of 100% acetone for 1 h at −20°C. The samples were centrifuged, and the pellet was resuspended in 80% (v/v) acetone and 10 mm DTT. Samples were recentrifuged, and the final pellets were dried with N2. IEF was performed in a vertical mini-gel format as described by Anderson and Peck (2008). Samples were solubilized in IEF sample buffer (7 m urea, 2 m thiourea, 2% [w/v] CHAPS, 0.8% [v/v] ampholytes, pH 3–10, 50 mm DTT, 4% [v/v] glycerol, and a trace of Bromophenol Blue) for 1 h and centrifuged to remove insoluble material. IEF gels (9 m urea, 1% [w/v] CHAPS, and 6% [v/v] polyacrylamide) were cast with 0.4% (v/v) broad-range ampholytes (pH 3–10) and subjected to electrophoresis using a Mini-Protean II apparatus (Bio-Rad) with 25 mm Tris and 10 mm phosphoric acid in the upper and lower chambers, respectively. For separation of proteins in the second dimension, single lanes were excised from the focused gels and incubated with equilibration buffer (60 mm Tris, pH 6.8, 2% [w/v] SDS, 0.1 m DTT, and 10% [v/v] glycerol) for 10 min. Gel pieces were placed across 12% (w/v) SDS-PAGE gels and overlaid with equilibration buffer supplemented with 1% (w/v) agarose and a trace of Bromophenol Blue. Second dimension gels were incubated with 12% (v/v) TCA and stained with a modified Coomassie Blue stain (27% [v/v] ethanol, 10% [v/v] acetic acid, 0.08% [w/v] Coomassie Blue R-250, and 0.5% [w/v] CuSO4) to reduce background staining of ampholytes or immunoblotted as described above. Low-Temperature (77 K) Fluorescence Samples of purified thylakoids and complex fractions were pipetted into NMR tubes, frozen in liquid nitrogen, and excited at 436 nm (QuantaMaster; Photon Technology International Canada), and emission spectra were collected between 650 and 800 nm using the 814 Photomultiplier Detection System fluorometer (Photon Technology International Canada) with Felix32 Analysis Module (version 1.2) software (Photon Technology International Canada). A slit width of 4 nm was used for both excitation and emission. Chlorophyll concentration ranged from 5 to 12 μg mL−1, and spectra were normalized to a maximum signal value of 1 for comparison. All spectra were corrected for the wavelength dependence of the detector. Spectrophotometric Measurements Spectroscopic assay of Cyt f in the purified PSI supercomplex fraction of UWO 241 was performed by absorbance difference spectrum (500–600 nm) of the reduced (3 mm hydroquinone) minus oxidized (1 mm potassium ferricyanide) absorption spectra as described (Wasserman, 1980; Greene et al., 1988). The spectra were recorded at room temperature using a Cary 50 Bio UV-VIS spectrophotometer (Varian Instruments). The α peak of Cyt f was defined with respect to the isosbestic points of 535 and 565 nm. Measurements of P700 Photooxidation FR light-induced photooxidation of P700 in SAG 49.72 and UWO 241 was estimated as ƊA 820-860 using the PAM-101 chlorophyll fluorescence measuring system equipped with dual-wavelength emitter-detector ED-P700DW and PAM-102 units (Heinz Walz) as described in detail earlier (Klughammer and Schreiber, 1991; Ivanov et al., 2000). Samples were prepared as described by Herbert et al. (1995). The relative redox state of P700 was determined in vivo under ambient CO2 conditions at the corresponding growth temperature of 5°C (UWO 241) or 25°C (SAG 49.72). FR light (λmax = 715 nm, 10 W m−2; Schott filter RG 715) was provided by an FL-101 light source. After reaching a steady-state level of P700+ in the presence of FR light background, single turnover (half peak width of 14 µs) and multiple turnover (50 ms) pulses of AL were applied with XMT-103 and XST-103 power/control units, respectively, via a multibranched fiber-optic system connected to the emitter-detector unit and the cuvette. Alternatively, P700 photooxidation was monitored in the presence of white AL exciting both PSI and PSII as described by Ivanov et al. (2012). White AL (150 μmol photons m−2 s−1) was provided by a Schott lamp (KL 1500; Schott Glaswerke) and controlled from a Heinz Walz PAM-103 trigger control unit. The P700 transients were measured in the absence and presence of the following inhibitors of electron transport: 40 µm DCMU and 20 µm AA. Freshly prepared stock solutions of DCMU and AA in 95% (v/v) ethanol were added to the cell suspension to a final solvent concentration of 0.5% (v/v). Samples were preincubated for 15 min in the dark at the corresponding growth temperatures in either the presence or absence of DCMU and AA prior to the measurement. All samples for P700 determinations were standardized on an equal chlorophyll basis, which allowed direct comparisons of the different growth conditions and treatments. Data analysis was performed using the Microcal Origin version 7.0 software package (Microcal Software). Sample Preparation for Nano-LC-ESI-MS/MS In-gel digestion was performed using a MassPREP automated digester station (Perkin-Elmer). Gel pieces were Coomassie Blue destained using 50 mm ammonium bicarbonate and 50% (v/v) acetonitrile, followed by protein reduction using 10 mm DTT, alkylation using 55 mm iodoacetamide, and tryptic digestion. Peptides were extracted using a solution of 1% (v/v) formic acid and 2% (v/v) acetonitrile and lyophilized. Prior to mass spectrometric analysis, dried peptide samples were redissolved in water and 0.1% (v/v) formic acid for liquid chromatography-tandem mass spectrometry analysis. Nano-LC-ESI-MS/MS Analysis Between 50% and 100% of each original sample was injected on a NanoAcquity ultra-performance liquid chromatograph (Waters) equipped with a 25-cm × 75-μm C18 reverse-phase column employing a 60-min liquid chromatography gradient (5%–40% [v/v] acetonitrile and 0.1% [v/v] formic acid) and detected in a data-dependent acquisition mode by tandem mass spectrometry (Q-ToF Ultima; Waters). The mass spectrometry was directed to use the following data-dependent acquisition parameters: survey scans range of 300 or 400 to 1,800 mass-to-charge ratio, 1-s scans, and one to four precursors selected based on charge state (+2, +3, and +4 ions). Tandem mass spectrometry fragmentation was then performed on the ions using the charge-state collision energy profile function. Raw data were converted to .pkl format using PLGS 2.2.5 (Protein Lynx Global Server; Waters). The resulting .pkl file was processed by PEAKS (BSI) for de novo sequencing and homology searching against the National Center for Biotechnology Information green plants database. The following settings were used: mass error of 0.15 D, fixed mode for carbamidomethyl Cys, variable mode for oxyM, Phospho STY, and Iodo Y. FDR values were calculated using the Decoy-Fusion method in PEAKS. Phosphorylated residues were identified by nano-performance liquid chromatography (Waters nAcquity) and tandem mass spectrometry (Thermo Orbitrap Elite Velos Pro) in the FT/IT/CID mode. Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Identification of SDS-solubilized subunits. Supplemental Figure S2. Absorption and 77K fluorescence emission spectra. Supplemental Figure S3. Identification of PSII phosphoproteins. Supplemental Figure S4. Heme staining of thylakoid membrane complexes. Supplemental Figure S5. Identification of phosphorylated tyrosine. Supplemental Figure S6. Comparison of sequence alignments. Supplemental Table S1. Blast results for protein spots 1 and 2. Supplemental Table S2. Peaks results for protein spot 3. Supplemental Table S3. Peaks results for protein spot 4. Supplemental Table S4. Peaks results for the PSI super complex. Glossary Chl a/b chlorophyll a/b CEF cyclic electron flow Cyt b 6/f cytochrome b 6/f Cyt f cytochrome f BN-PAGE blue-native PAGE DDM n-dodecyl-β-d-maltoside Chl a chlorophyll a IEF isoelectric focusing nano-LC-ESI-MS/MS liquid chromatography isoelectric focusing nano-tandem mass spectrometry Nano-LC-ESI-MS/MS liquid chromatography isoelectric focusing nano-tandem mass spectrometry FR far-red ƊA 820-860 absorbance changes at 820 nm AA antimycin A FQR ferredoxin-PQ reductase AL actinic light DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea BBM Bold’s basal medium DTT dithiothreitol LITERATURE CITED Allen JF ( 2003 ) State transitions: a question of balance . Science 299 : 1530 – 1532 Google Scholar Crossref Search ADS WorldCat Allen JF , Bennett J, Steinback KE, Arntzen CJ ( 1981 ) Chloroplast protein phosphorylation couples plastoquinone redox state to distribution of excitation energy between photosystems . 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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: Norman P.A. Hüner (nhuner@uwo.ca). B.S.-M. performed all the experiments and wrote the initial draft of the article; P.P. and G.L. performed the protein sequence analyses and edited the article; A.G.I. performed the P700 measurements and edited the article; N.P.A.H. and B.S.-M. conceived and planned all the experiments; N.P.A.H. also edited the article extensively. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00625 © 2015 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2015. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b  6/f Supercomplex  
JF - Plant Physiology
DO - 10.1104/pp.15.00625
DA - 2015-09-04
UR - https://www.deepdyve.com/lp/oxford-university-press/the-antarctic-psychrophile-chlamydomonas-sp-uwo-241-preferentially-LpnGHltvPN
SP - 717
EP - 736
VL - 169
IS - 1
DP - DeepDyve
ER -