TY - JOUR
AU1 - Rissler, Heather M.
AU2 - Durnford, Dion G.
AB - Abstract Novel eukaryotic chlorophyll–carotenoid proteins have evolved at least twice following the origin of the plastid and include the widely distributed integral membrane light-harvesting complexes (LHCs) and the dinoflagellate-specific soluble peridinin–chlorophyll proteins. In the glaucophytes, homologs of these proteins are reportedly absent. We have identified a novel carotenoid-rich protein (CRP) in the glaucophyte Cyanophora paradoxa that is 28 kDa and immunologically related to the family of LHCs. CRP is associated with the thylakoid membrane, though it can be removed by stringent washes, suggesting that there are probably significant structural differences between CRP and the LHCs. CRP co-localizes with a zeaxanthin-rich thylakoid membrane fraction that also contains β-carotene, chlorophyll and an unidentified carotenoid. Despite this, we found no evidence for carotenoid–chlorophyll energy transfer in the isolated complex, suggesting that light harvesting may not be a primary function. The presence of CRP in C. paradoxa is evidence for the evolution of a novel pigment-binding protein in the glaucophytes. Introduction Photosynthetic organisms use light-harvesting antennae to optimize the effective cross-sectional area for absorption of light energy and to regulate transfer of excitation energy to the photosynthetic reaction centers. In oxygenic photosynthetic organisms, several diverse classes of proteins that perform the function of light harvesting have evolved independently (Green and Durnford 1996). Light-harvesting antennae are comprised of pigment-binding proteins and their associated chromophores, which include tetrapyrroles and carotenoids. Both soluble and integral membrane proteins have evolved the function of light harvesting, and both types are ubiquitous amongst photosynthetic organisms. The antennae of most cyanobacteria are comprised of linear tetrapyrroles that are covalently linked to soluble proteins and assembled into phycobilisomes (PBS). Remnants of the cyanobacterial antennae system have persisted in several groups of algae including the rhodophytes and glaucophytes, which contain fully assembled PBS, and the cryptomonads, which possess phycobiliproteins. A second type of independently evolved light-harvesting antennae, the soluble peridinin–chlorophyll-binding protein (PCP), is present in many dinoflagellates (Prezelin and Haxo 1976). The most widespread class of antennae proteins found in algae and higher plants are the light-harvesting complexes (LHCs), which have three membrane-spanning regions (MSRs) that non-covalently bind chlorophylls (Chls) and carotenoids (Green and Durnford 1996). Evolution of the LHC gene family has produced an impressive array of apoproteins that can bind a range of accessory pigments, enabling photosynthetic organisms to optimize light utilization in diverse environments (Durnford et al. 1999). While light harvesting is an important function of pigment-binding proteins, many photoprotective proteins have also evolved independently. In particular, many members of the LHC superfamily are hypothesized to serve a photoprotective function and can be categorized based on the number of predicted MSRs. The high light-inducible proteins (HLIPs) in cyanobacteria possess one MSR (Bhaya et al. 2002), and HLIP homologs are also found in eukaryotes (reviewed in Heddad and Adamska 2002). Stress-enhanced proteins (SEPs), which posses two MSRs, are induced in response to light stress in Arabidopsis thaliana (Heddad and Adamska 2000). The family of early light-induced proteins (ELIPs) possess three MSRs and are important in mediating defense against photo-oxidative stress (Hutin et al. 2003). Finally, the PsbS protein found in higher plants has four predicted MSRs (Funk et al. 1995) and is essential for non-photochemical quenching of Chl fluorescence (Li et al. 2000). Soluble pigment–protein complexes with photoprotective properties ranging from carotenoid-mediated quenching of Chl fluorescence to provision of a pigment–protein environment that prevents reaction of Chl with triplet oxygen have also evolved independently. Within this group are the soluble carotenoid proteins of cyanobacteria (Wu and Krogmann 1997) and the water-soluble chlorophyll protein (WSC) found in higher plants (Satoh et al. 2001). It is widely accepted that plastids of algae and plants arose from an endosymbiotic association between a cyanobacterium and a eukaryotic host (Delwiche 1999). Of the three separate algal groups believed to have obtained plastids via a single primary endosymbiotic event—red algae, green algae and glaucophytes—only the red and green algae are known to posses LHCs. The presence of LHCs in these divisions to the exclusion of cyanobacteria supported a monophyletic origin of the primary plastids (Wolfe et al. 1994). The glaucophytes have long been regarded as an evolutionary oddity due to the presence of a remnant peptidoglycan wall surrounding the plastid. Though the plastid clearly evolved from a cyanobacterium, they often form only a weak sister group to the red and green algal lineages (Moreira et al. 2000); thus the origin of their plastids has not been convincingly resolved. A study using two different antisera to LHCs failed to find any immunological evidence for their presence in the glaucophyte, Cyanophora paradoxa (Koike et al. 2000), which suggests that LHCs either evolved following the divergence of glaucophytes from the red and green algal lineages, or that there was an independent plastid acquisition event. To resolve the evolution of the eukaryotic LHCs further, we examined the pigment–protein complexes in C. paradoxa. We have identified a pigment-binding protein in C. paradoxa, carotenoid-rich protein (CRP), which is immunologically related to the LHCs but with different biochemical properties. The potential role of CRP in C. paradoxa and its implication for the evolution of antennae complexes will be discussed. Results C. paradoxa possesses a pigment-binding protein that is associated with thylakoid membranes and is immunologically related to LHCs Using an antibody (anti-FCP) derived against a fucoxanthin–chlorophyll protein from Heterosigma carterae, a marine raphidophyte, we detected a 28 kDa protein in whole-cell extracts that we will refer to as CRP for carotenoid-rich protein (Fig. 1A). Anti-FCP also cross-reacts with LHCs from both red and green algae, detecting at least three apoproteins of photosystem II (PSII) in the green alga Chlamydomonas reinhardtii (25, 30 and 33 kDa) and two LHCI apoproteins in the red alga Rhodomela confervoides (14 and 18 kDa) (Fig. 1B, D), while no immunologically related bands were detected in the cyanobacterium Synechococcus PCC7942 (Fig. 1C). To determine the subcellular localization of CRP, cyanelles were isolated and separated into soluble and membrane fractions. The efficiency of separation was verified by analyses of pigment and protein contents of the two fractions. The cyanelle membrane fraction contained all of the Chl a and carotenoids and only trace amounts of PBS chromophores relative to the soluble fraction (Fig. 2A). Immunoblot analyses of membrane and soluble fractions with anti-FCP and anti-D1 showed that CRP is localized to the thylakoid membrane fraction of cyanelles (Fig. 2B). Unexpectedly, CRP appears to be only tightly associated with thylakoid membranes as it is partially removed by high-salt washes (Fig. 2C) unlike the LHCs of Chlamydomonas that remained in the membrane fraction (data not shown). To explore the properties of the CRP further, we used the technique of phase partitioning with Triton X-114, which separates integral and peripheral membrane proteins (Bordier 1981). Following phase partitioning, CRP was retained predominantly in the soluble phase, supporting the observation that it is easily dissociated from the membrane (Fig. 2D). D1, a known integral membrane protein, was present in the soluble and membrane phase, a phenomenon that has been reported previously for thylakoid membrane proteins (Zak et al. 1999) (Fig. 2D). Cyanelle membranes were also extracted with chloroform : methanol (5 : 4, v/v), a procedure that is able to solubilize hydrophobic membrane proteins. When cyanelle membrane proteins were extracted with this organic solvent, CRP appeared in the insoluble fraction while D1 was found in the chloroform : methanol-soluble phase (Fig. 2E). This indicated that CRP is not very hydrophobic and if it is a membrane protein there is probably a large soluble portion. When cyanelle membranes were treated with trypsin, the CRP size was unaltered, in contrast to the LHCs of Chlamydomonas that were partially digested (Fig. 2F). CRP remained intact following 20 min of trypsin treatment (data not shown), suggesting that its association with the membrane affords protection against proteolytic damage. CRP is associated predominantly with zeaxanthin To characterize further the biochemical properties of CRP, cyanelle membranes from C. paradoxa were fractionated by sucrose density centrifugation following β-d-maltopyransoside solubilization, resulting in two predominant pigmented fractions: an upper yellow fraction (YF) and a lower green fraction (GF) (Fig. 3A). Proteins from both fractions were analyzed by SDS–PAGE, and Western blot analyses demonstrated the presence of the PSII reaction center protein D1 in the GF (Fig. 3B). Though there were traces of the CRP in the GF, it was very abundant in the YF (Fig. 3B) and significantly enriched on a per protein basis relative to thylakoid membranes. D1 was absent in the YF, suggesting that very little cross-contamination occurred between the two fractions. The absorbance spectrum of the GF was similar to that of whole thylakoid membranes (Fig. 3C). In contrast, the YF was rich in carotenoids and contained a smaller amount of Chl (Fig. 3D). The molar ratios of carotenoids to Chl in the GF and YF were 0.23 ± 0.04 and 2.3 ± 0.6, respectively. The predominant carotenoids that accumulate in C. paradoxa are zeaxanthin, β-carotene (Chapman 1966) and β-cryptoxanthin in small quantities (Schmidt et al. 1979), and pigments in the YF were separated by thin-layer chromatography (TLC) to determine the proportion of each (Fig. 4). The major pigment component of the YF was identified as zeaxanthin (orange-yellow band, rf = 0.59) based on previous studies (Chapman 1966, Schmidt et al. 1979) and its absorption maxima in ethanol (Davies and Köst 1988). Chl a (green band, rf = 0.73) and β-carotene (orange band, rf = 0.98) were also present in this fraction (Fig. 4). A thin orange band running near the front of the Chl a band was visible on the TLC plate. This pigment may be β-cryptoxanthin as discussed by Schmidt et al. (1979). The presence of a carotenoid is obvious in the spectra of the green (Chl a) band of the YF, which has the unexpected shoulder from 450 to 500 nm (Fig. 4). This carotenoid was not detected in the Chl band from the GF, suggesting that it is exclusively associated with the CRP. Though there was variability in the pigment content of the YF, probably due to the labile nature of the complex, the molar ratios of the pigments on average were: 1 Chl a; 0.2 ± 0.02 unknown carotenoid (‘β-cryptoxanthin’); 0.5 ± 0.4 β-carotene; 3.2 ± 1.1 zeaxanthin. The free pigment (FP) fraction at the top of the gradient was composed almost entirely of β-carotene with a trace amount of Chl a only visible under UV light (data not shown). When proteins from the YF were purified further by anion exchange chromatography, a pigmented band containing both Chls and carotenoids was eluted from the column following a wash with 0.5 M NaCl (Fig. 5A). Western blot analyses verified that the pigmented band was enriched in CRP, further verifying the association of this protein with pigments (Fig. 5B). Free pigment consisting primarily of carotenoid was eluted from the column with salt-free wash buffer (Fig. 5A) indicative of the labile nature of this pigment–protein. A similar carotenoid-rich fraction was detected by an independent biochemical technique that fractionated detergent-solubilized thylakoid membranes by non-denaturing gel electrophoresis. Three pigmented bands were resolved in C. paradoxa thylakoid membranes (P1–P3) in addition to a FP band comprised predominantly of carotenoids (Fig. 6A). As expected, the higher molecular weight bands containing photosynthetic reaction centers lacked auto-fluorescence upon exposure to UV light, in contrast to P3 (Fig. 6A). SDS–PAGE analyses of proteins eluted from band P3 indicated it was enriched in a 28 kDa protein that was absent in P1 and P2 (Fig. 6B). Western blot analyses of proteins eluted from P2 showed an enrichment of D1, suggesting that P2 corresponds to PSII complexes. P3 was enriched in CRP (Fig. 6C). Though the YF absorbs light strongly in the 450–520 nm region due to the abundance of carotenoids, there was little evidence for efficient transfer of excitation energy directly to chlorophyll when the complex was excited at 487 nm, compared with 436 nm where Chl absorbs strongly (Fig. 7, top panel). The small amount of emission at 680 nm when excited at 487 nm appears to be residual Chl absorption, as boiling the sample led to similar, small declines in fluorescence yield at both excitation wavelengths (data not shown). Emission at 680 nm was significant only when Chl was directly excited (<450 nm) (Fig. 7). Since this complex was purified with detergents, energy transfer could have been disrupted, therefore we conducted similar measurements on purified thylakoid membranes (Fig. 7, lower panel). In thylakoids, excitation at 487 nm still did not yield evidence for efficient energy transfer from carotenoid to Chl, despite the abundance of carotenoids in this fraction. As with the YF, Chl emission at 680 nm was generated primarily by wavelengths where Chl absorbs maximally. The increase in fluorescence emission in thylakoids when excited at >510 nm is due to the absorption of PBS that remained attached during purification of thylakoid membranes. CRP accumulation is down-regulated in response to high light To determine whether the co-localization of CRP with the carotenoid-rich fraction of thylakoid membranes may be indicative a photoprotective function, we investigated the regulation of CRP accumulation in response to high light (HL). Photoacclimation was initiated by a 10-fold increase in growth irradiance, and changes in CRP accumulation in C. paradoxa were monitored after 24 h. The maximum yield of PSII (FV/FM) was reduced by 20% in HL-grown cultures, indicating that this was a significant light stress (Table 1). Following a 24 h exposure to HL, Chl a content per cell increased slightly although the amount of Chl per protein in thylakoid membranes was reduced by 14% (Table 1). The differences in pigment composition on a per cell basis can be attributed to changes in the density of thylakoid membrane components in HL-acclimated cells, rather than an increase in concentration of Chl-binding proteins in the thylakoid membranes. Total carotenoid content per cell, however, increased 50% following a shift into HL (Table 1). Fractionation of thylakoid membranes revealed a 5-fold increase in the amount of FP isolated from HL-exposed cultures (Table 1) that is comprised primarily of β-carotene as determined by TLC (data not shown). Therefore, the increase in carotenoids in HL-acclimated C. paradoxa is likely to be accounted for by an increase in membrane-localized β-carotene, as opposed to carotenoids associated with pigment–protein complexes, such as the CRP. Photosynthetic irradiance response curves for low light (LL)- and HL-acclimated cultures show a light saturation point for O2 evolution of 100 and 300 µmol photons m–2 s–1, respectively (Table 1). The requirement for a higher light intensity to saturate photosynthesis in HL-acclimated cultures suggests a down-regulation in light-harvesting antennae size, a phenomenon that is a common response of photosynthetic organisms to increases in irradiance levels (Anderson et al. 1995). In support of this reduced light-harvesting capacity, there was a decrease in PBS fluorescence following HL exposure (Fig. 8A) that was paralleled by a reduction in CRP content during this same period (Fig. 8B). The concentration of PSII reaction centers per cell also declined following HL exposure (Fig. 8C). Discussion Identification and characterization of a novel pigment-binding protein in C. paradoxa Using an antibody derived against an FCP, we were able to detect a thylakoid-localized protein in C. paradoxa that is immunologically related to LHCs. The labile nature of its association with the thylakoid membrane (Fig. 2), however, is quite unusual given its immunological relatedness to a pigment-binding protein with three MSRs. Different extraction procedures on cyanelle membranes suggest that this membrane protein is not very hydrophobic and is easily liberated from its association with the thylakoid membrane. The location of CRP in the thylakoid membrane offers it protection from trypsin digests, indicating that any loop or soluble regions are not accessible due to its association with the membrane or other proteins. Though the cross-reactivity of the CRP with the FCP antisera indicates the presence of shared epitopes, there are likely to be significant structural differences between it and the LHC gene family. First, although CRP cross-reacts with the FCP antisera, it only cross-reacts very weakly with a Chlamydomonas LHC-specific antiserum (data not shown), or not at all, as was experienced by another group (Koike et al. 2000). This indicates that the CRP and the LHCs share relatively few antigenic epitopes, and the immunoreactivity to the FCP antiserum could be explained by the presence of pigment-binding motifs that are specific to the FCPs. Second, the CRP can be partially dissociated from thylakoid membranes, suggesting that it is not as hydrophobic as the LHC protein family. Although the LHC-related, LI818 protein of C. reinhardtii displays similar biochemical properties in that it can be partially removed by salt washes and alkaline treatments (Richard et al. 2000), we are not certain that CRP is a bona fide LHC. It is equally possible that a novel pigment-binding protein has evolved from small LHC-like proteins such as the HLIPs, which are present in Cyanophora. It is also conceivable that the CRP is not an LHC-related protein but that it only shares structural motifs that participate in pigment binding, that may account for the immunological similarity. One example of this is found in the WSC proteins, which share the [F/Y]DPLGL motif that is conserved in LHCs (Satoh et al. 2001). We are working towards sequencing the protein and cloning the gene to determine the basis of this cross-reactivity. Several biochemical techniques were used to examine the association of CRP with photosynthetic pigments. Separation of thylakoid membrane proteins by sucrose gradient fractionation, anion exchange chromatography and non-denaturing gel electrophoresis resulted in isolation of a CRP-enriched fraction containing zeaxanthin, chlorophyll, β-carotene and a small amount of another carotenoid (Fig. 3–5), tentatively identified as β-cryptoxanthin based on the work of Schmidt et al. (1979). Under these mild, non-denaturing conditions and during anion exchange chromatography, the bound zeaxanthin is easily dissociated, suggesting that pigments are non-covalently bound to CRP (Fig. 5A). The correlation of the immunoreactive 28 kDa band with a carotenoid-rich fraction in three independent fractionation procedures indicates that the co-migration of a non-specific band that cross-reacts with this antiserum is unlikely and that this protein actually shares epitopes with the FCP family of proteins. While the pigment-binding properties of LHCs are quite diverse, the molar ratio of carotenoids to Chls typically ranges from 0.5 to 1.3 in LHCs found in chlorophytes, rhodophytes and chromophytes (Hiller et al. 1993, Bassi and Caffarri 2000, De Martino et al. 2000, Grabowski et al. 2001). CRP, however, has a relatively high carotenoid to Chl ratio, which is often indicative of a photoprotective role rather than light harvesting, as is the case with the ELIPs of higher plants (Adamska et al. 1999) and the carotenoid biosynthesis-related (CBR) protein of Dunaliella salina (Banet et al. 2000). In all cases, exposure to HL stress leads to an induction of these proteins (Havaux et al. 2003). In contrast, LHCs of algae and higher plants that are involved in light harvesting are down-regulated in response to increased irradiance, as a smaller antennae size is necessary to absorb an efficient number of photons to drive photochemistry without over-excitation of the reaction centers (Anderson et al. 1995). During HL acclimation in C. paradoxa, the total carotenoid content increased on a per cell basis (Table 1), although the total amount of carotenoids localized to thylakoid membranes remained constant following HL acclimation (Table 2). An increase in ‘free carotenoids’ in HL-acclimated C. paradoxa is not surprising, given that such carotenoids have roles in preventing peroxidation of plastid membranes (Havaux 1998) and in the regulation of membrane fluidity (Ourisson and Nakatani 1995). Despite the increase in total cellular carotenoids, CRP accumulation was down-regulated in concert with PBS following HL acclimation. Although the decrease in CRP would suggest a role in light harvesting, we were unable to detect significant carotenoid to Chl energy transfer in the YF from the sucrose gradient or in thylakoid membranes. If not an antenna, then CRP is likely to participate in photoprotection or the regulation of energy distribution to the reaction centers by an unknown mechanism. Given the abundance of zeaxanthin, a role in energy dissipation is a strong possibility. The decline in HL indicates that CRP is not strictly associated with stress like the ELIPs and CBR protein of Dunaliella, the latter of which is also proposed to bind zeaxanthin (Levy et al. 1993). Instead, CRP is abundant under our LL conditions and thus has an important role in the ‘unstressed’ state. Although a decline in CRP abundance in HL seems to be counter to a proposed role in photoprotection, it may function in fixed stoichiometry with respect to reaction centers or PBS, which both decline under HL. CRP may represent a unique innovation in the regulation of light harvesting, although it is unclear at this stage whether CRP is involved in energy dissipation, regulating the transfer of excitation energy to reaction centers or if it has another role related to photoprotection or light harvesting. Further studies are necessary to understand the pigment-binding properties of CRP and its role in C. paradoxa. Elucidating the nature of light harvesting and photoprotective strategies employed by this enigmatic photosynthetic eukaryote will provide insight into the evolution and diversification of antennae systems that followed the acquisition of plastids. Materials and Methods Growth conditions and collection of algae Cyanophora paradoxa Korsh strain LB555 obtained from the Culture Collection of Algae (University of Texas at Austin) were grown in modified WCg medium buffered with 10 mM HEPES-NaOH (pH 7.5) bubbled with humidified air (Guillard and Ryther 1962, Guillard 1975). C. paradoxa cultures were grown to a maximum cell density of 1×106 cells ml–1 at 24°C in LL (55 µmol photons m–2 s–1). To induce a light stress, cultures were shifted to HL (500 µmol photons m–2 s–1) for 24 h. C. reinhardtii strain CC125 was grown in Tris-acetate-phosphate medium as described (Durnford et al. 2003). R. confervoides (Rhodophyta; Subclass Florideophycidae) was collected at Green’s Point (Letete, NB, Canada). Synechococcus PCC7942 was obtained from Dr. D. Campbell. Protein extraction from whole cells and isolated cyanelles For extraction of total cellular proteins from C. paradoxa, C. reinhardtii and Synechococcus PCC7942, 5×107 cells were harvested and proteins extracted in 50 µl of lysis solution containing 0.2 M Na2CO3, 2% SDS (w/v), 5 mM aminocaproic acid and 1 mM benzamidine HCl. R. confervoides tissue (500 mg) was frozen in liquid N2 and ground to a fine powder with a mortar and pestle and resuspended in 500 µl of lysis solution. The mixture was incubated at 60°C for 10 min. Following lysis, all samples were centrifuged at 14,000×g for 5 min and the resulting supernatant was used for protein analyses. Cyanelles were isolated from C. paradoxa (∼7×109 cells) and lysed as previously described (Koike et al. 2000). Lysed cyanelles were centrifuged at 150,000×g for 1 h at 4°C. The soluble blue supernatant was removed and used directly for further analyses. The pellet, containing crude thylakoid membranes, was resuspended in HEMS buffer [50 mM HEPES-NaOH (pH 7.5), 2 mM EGTA, 1 mM MgCl2 and 0.5 M sucrose] to a final protein concentration of 5 µg µl–1. Thylakoid membrane treatments To examine association of proteins with membranes, crude thylakoid membranes were prepared as described above and washed with 0.5 M NaCl in HEMS buffer for 20 min at 4°C followed by centrifugation at 150,000×g for 30 min. Supernatants were collected and concentrated by precipitation in 90% (w/v) acetone and membranes were resuspended in HEMS buffer. Protein concentrations were determined using the BCA protein assay (Pierce; Rockford, IL, U.S.A.). Cyanelle membranes were subjected to Triton X-114 phase partitioning as previously described (Bordier 1981) and also extracted in chloroform : methanol [5 : 4 (v/v)] according to Ferro et al. (2000). Thylakoid membrane fractionation and anion exchange chromatography Crude thylakoid membranes isolated from C. paradoxa were resuspended in HEMS buffer to a final Chl concentration of 0.5 (mg Chl) ml–1 and solubilized with 1% β-d-maltopyransoside (w/v) for 10 min at 4°C. The suspension was centrifuged at 8,600×g for 5 min at 4°C, and the supernatant placed on a linear sucrose gradient [5-30% (w/v) sucrose in HEMS buffer] and centrifuged at 150,000×g for 14 h at 4°C. Sucrose gradients were created using the Gradient Master (BioComp Inst. NB, Canada). Colored fractions were removed from the gradients and concentrated using a 5 kDa cut-off centrifugal filter unit prior to further analyses (Millipore; Billerica, MA, U.S.A.). Concentrated sucrose gradient fractions were loaded onto a column containing DEAE anion exchange resin (Amersham Pharmacia; Uppsala, Sweden) pre-equilibrated with 50 mM HEPES and 0.01% β-d-maltopyransoside (w/v). Washes were performed with 10 and 500 mM NaCl. Proteins were precipitated from collected wash fractions with 100% (w/v) acetone and resuspended in HEMS buffer. Non-denaturing gel electrophoresis Following detergent solubilization of thylakoid membranes, as described above, samples containing 15 µg of Chl were separated under non-denaturing conditions on 8% (w/v) polyacrylamide gels (Allen and Staehelin 1991). Pigmented bands were excised from the gel and proteins were eluted by incubation at 90°C for 15 min in sample loading buffer [125 mM Tris–HCl, pH 6.8; 2% (w/v) SDS; 2% (w/v) β-mercaptoethanol; 0.05% (w/v) bromophenol blue, 20% (v/v) glycerol]. SDS–PAGE and immunoblot analyses Protein samples were separated by electrophoresis on 14% Tris–glycine, SDS–polyacrylamide gels (Sambrook et al. 1989). For comparisons of protein levels between light regimes, samples were loaded on an equal cell basis (equivalent to protein extracted from 1.9×106 cells). For cross-species comparisons and all other analyses, proteins were loaded on an equal protein basis (20 µg). To compare relative levels of PBS, gels were stained with 75 mM ZnSO4 (Raps 1990). For detection of proteins following SDS–PAGE, gels were subjected to silver staining as previously described (Oakley et al. 1980). Immunoblot analyses were done as previously described (Durnford et al. 2003). Antiserum directed against the core reaction center of PSII (D1) was provided by Dr. D. Campbell. The anti-FCP antiserum was made against the FCPs of a marine chromophyte (Heretosigma carterae; Harnett 1998) and was provided by Dr. B.R. Green. Pigment extraction, thin layer chromatography and quantification Chls and carotenoids were extracted from cell pellets and the pigment concentrations were determined using previously published molar extinction coefficients (Lichtenthaler 1987, Tandeau de Marsac and Houmard 1988). Absorbance spectra were measured with a Cary 100 spectrophotometer (Varian, Palo Alto, CA, U.S.A.). Sucrose gradient fractions were extracted in 100% acetone followed by petroleum ether. The petroleum ether layer containing the pigments was removed and the pigments concentrated by evaporation under N2 gas and spotted on a silica gel 60 TLC plate (0.25 mm thickness). Pigments were separated with petroleum ether : isopropanol : water mixture (100 : 10 : 0.25, by vol.) in the dark. Bands containing pigments were scraped off the plate and eluted in 95% ethanol (carotenoids) or 80% acetone (Chl) and quantified as described above. Oxygen evolution and fluorescence measurements Cyanophora paradoxa cells were concentrated to a cell density of 8×106 cells ml–1 for oxygen evolution and fluorescence measurements. Oxygen evolution and the maximum efficiency of PSII (FV/FM) were measured as described previously (Durnford et al. 2003). Room temperature fluorescence and excitation measurements were conducted on a Perkin-Elmer LS50 spectrophotometer equipped with a red-sensitive photomultiplier. Samples were diluted in HEMS to a concentration of approximately 1–2 µg ml–1 prior to measuring. Acknowledgments We are grateful to Drs. B.R. Green, D. Campbell and K. Hoober for providing the FCP, D1, and Chlamydomonas LHC antisera, respectively. We thank Dr. G. Saunders for assisting with the collection of Rhodomela confervoides, and Dr. V. Erickson and I. Erickson for assistance in translating German. This work was supported by a grant from the National Sciences and Engineering Council of Canada. H.R. is funded under the auspices of PEP, the Protist EST Program, which is funded by Genome Canada, Genome Atlantic and the Atlantic Innovation Fund. View largeDownload slide Fig. 1 Immunoblot of total cellular proteins from (A) the green alga Chlamydomonas reinhardtii, (B) the red alga Rhodomela confervoides, (C) the cyanobacterium Synechococcus PCC7942 and (D) the glaucophyte Cyanophora paradoxa. Lanes were normalized to total cell protein per lane. Proteins were separated by SDS–PAGE and visualized by Western blot analyses with an antibody raised to the fucoxanthin–Chl protein of a marine raphidophyte (anti-FCP). View largeDownload slide Fig. 1 Immunoblot of total cellular proteins from (A) the green alga Chlamydomonas reinhardtii, (B) the red alga Rhodomela confervoides, (C) the cyanobacterium Synechococcus PCC7942 and (D) the glaucophyte Cyanophora paradoxa. Lanes were normalized to total cell protein per lane. Proteins were separated by SDS–PAGE and visualized by Western blot analyses with an antibody raised to the fucoxanthin–Chl protein of a marine raphidophyte (anti-FCP). View largeDownload slide Fig. 2 (A) Proteins from whole cells (WC), cyanelle membranes (M) and soluble cyanelles proteins (S) were separated by SDS–PAGE. Phycobilisomes (PBS) were visualized by enhanced fluorescence following staining with ZnSO4. (B) CRP and D1 were visualized by Western blot analyses with anti-FCP and anti-D1 (an antibody derived from the D1 protein of PSII). (C) Thylakoid membrane proteins (M), membranes washed with 0.5 mM NaCl (WM) and proteins removed by washing with 0.5 mM NaCl (Su) were visualized by Western blot analyses with anti-FCP and anti-D1 antisera. (D) Cyanelle membrane proteins were separated by Triton X-114 phase partitioning into membrane (TrM) and soluble (TrS) phases. Proteins were visualized by Western blot analyses with anti-FCP and anti-D1. (E) Thylakoid membranes were either untreated (Th) or extracted with chloroform : methanol (5 : 4 v/v) and separated into soluble (S) and insoluble (In) fractions. The presence of D1 and CRP in each of these fractions was determined by Western blot using the anti-D1 and anti-FCP antisera (F) Thylakoid membranes from C. reinhardtii (C.r.) and C. paradoxa (C.p.) were incubated without (–T) or with (+T) trypsin and analyzed by Western blot analysis with anti-FCP. View largeDownload slide Fig. 2 (A) Proteins from whole cells (WC), cyanelle membranes (M) and soluble cyanelles proteins (S) were separated by SDS–PAGE. Phycobilisomes (PBS) were visualized by enhanced fluorescence following staining with ZnSO4. (B) CRP and D1 were visualized by Western blot analyses with anti-FCP and anti-D1 (an antibody derived from the D1 protein of PSII). (C) Thylakoid membrane proteins (M), membranes washed with 0.5 mM NaCl (WM) and proteins removed by washing with 0.5 mM NaCl (Su) were visualized by Western blot analyses with anti-FCP and anti-D1 antisera. (D) Cyanelle membrane proteins were separated by Triton X-114 phase partitioning into membrane (TrM) and soluble (TrS) phases. Proteins were visualized by Western blot analyses with anti-FCP and anti-D1. (E) Thylakoid membranes were either untreated (Th) or extracted with chloroform : methanol (5 : 4 v/v) and separated into soluble (S) and insoluble (In) fractions. The presence of D1 and CRP in each of these fractions was determined by Western blot using the anti-D1 and anti-FCP antisera (F) Thylakoid membranes from C. reinhardtii (C.r.) and C. paradoxa (C.p.) were incubated without (–T) or with (+T) trypsin and analyzed by Western blot analysis with anti-FCP. View largeDownload slide Fig. 3 (A) Thylakoid membranes of C. paradoxa were treated with 0.1% β-d-maltopyranoside and fractionated by sucrose density centrifugation, resulting in three fractions (depicted diagrammatically): a minor, free pigment (FP) fraction, an upper yellow fraction (YF) and a lower green fraction (GF). (B) Proteins from whole (Th) and fractionated thylakoid membranes were separated by SDS–PAGE and visualized by Western blot analyses with anti-FCP and anti-D1 antisera. (C and D) Absorbance spectra in 90% methanol are shown for the upper YF (C) and for the lower green fraction (D). The absorbance spectrum of thylakoid membranes is shown as a reference. View largeDownload slide Fig. 3 (A) Thylakoid membranes of C. paradoxa were treated with 0.1% β-d-maltopyranoside and fractionated by sucrose density centrifugation, resulting in three fractions (depicted diagrammatically): a minor, free pigment (FP) fraction, an upper yellow fraction (YF) and a lower green fraction (GF). (B) Proteins from whole (Th) and fractionated thylakoid membranes were separated by SDS–PAGE and visualized by Western blot analyses with anti-FCP and anti-D1 antisera. (C and D) Absorbance spectra in 90% methanol are shown for the upper YF (C) and for the lower green fraction (D). The absorbance spectrum of thylakoid membranes is shown as a reference. View largeDownload slide Fig. 4 Absorption spectra of the three pigments isolated following TLC separation of the YF from the sucrose gradient. The orange-yellow band (λmax 452, 479) and orange bands (λmax 450) were dissolved in 95% ethanol, while the green band was eluted in 80% acetone (λmax 415, 663). The orange-yellow band on the TLC plate was yellow when eluted in 95% ethanol. View largeDownload slide Fig. 4 Absorption spectra of the three pigments isolated following TLC separation of the YF from the sucrose gradient. The orange-yellow band (λmax 452, 479) and orange bands (λmax 450) were dissolved in 95% ethanol, while the green band was eluted in 80% acetone (λmax 415, 663). The orange-yellow band on the TLC plate was yellow when eluted in 95% ethanol. View largeDownload slide Fig. 5 Yellow fractions (YFs) obtained from sucrose density centrifugation of C. paradoxa thylakoid membranes (Th) were separated further by anion exchange chromatography. (A) Absorbance spectra of the flow-through (F/T, solid line) and the pigmented band (Ax, dashed line) obtained following a 0.5 M NaCl wash are shown. (B and C) Proteins were separated by SDS–PAGE and visualized by Western blotting with anti-FCP (B) and silver staining (C). CRP was not detected in the F/T fraction (data not shown). View largeDownload slide Fig. 5 Yellow fractions (YFs) obtained from sucrose density centrifugation of C. paradoxa thylakoid membranes (Th) were separated further by anion exchange chromatography. (A) Absorbance spectra of the flow-through (F/T, solid line) and the pigmented band (Ax, dashed line) obtained following a 0.5 M NaCl wash are shown. (B and C) Proteins were separated by SDS–PAGE and visualized by Western blotting with anti-FCP (B) and silver staining (C). CRP was not detected in the F/T fraction (data not shown). View largeDownload slide Fig. 6 (A) Thylakoid membranes from C. paradoxa were solubilized with 1% (w/v) β-d-maltopyranoside and separated by non-denaturing gel electrophoresis on an 8% (w/v) polyacrylamide gel. Pigmented protein bands (P1–P3) and the free pigment band (FP) are indicated. Gels were exposed to UV light in order to visualize fluorescent bands (right panel). Pigmented bands were excised from non-denaturing polyacrylamide gels and proteins were eluted. (B) Proteins were separated by SDS–PAGE and visualized by silver staining. (C) Western blot analyses with anti-FCP. An abundant 28 kDa protein detected by silver staining in P3 may correspond to the protein exhibiting immunoreactivity with anti-FCP (CRP). View largeDownload slide Fig. 6 (A) Thylakoid membranes from C. paradoxa were solubilized with 1% (w/v) β-d-maltopyranoside and separated by non-denaturing gel electrophoresis on an 8% (w/v) polyacrylamide gel. Pigmented protein bands (P1–P3) and the free pigment band (FP) are indicated. Gels were exposed to UV light in order to visualize fluorescent bands (right panel). Pigmented bands were excised from non-denaturing polyacrylamide gels and proteins were eluted. (B) Proteins were separated by SDS–PAGE and visualized by silver staining. (C) Western blot analyses with anti-FCP. An abundant 28 kDa protein detected by silver staining in P3 may correspond to the protein exhibiting immunoreactivity with anti-FCP (CRP). View largeDownload slide Fig. 7 Room temperature fluorescence characteristics of the yellow fraction (top panel) and thylakoid membranes (bottom panel). The excitation spectrum (em680, emission at 680 nm) and emission spectra excited at either 436 nm (ex436) or 487 nm (ex487) were determined for the YF from both the sucrose gradient (top panel) and thylakoid membranes (bottom panel). The absorbance spectra for each are provided (dashed lines). The absorbance spectrum of the soluble phycobilisome fraction (PBS Abs) is also provided for comparison in the bottom panel. View largeDownload slide Fig. 7 Room temperature fluorescence characteristics of the yellow fraction (top panel) and thylakoid membranes (bottom panel). The excitation spectrum (em680, emission at 680 nm) and emission spectra excited at either 436 nm (ex436) or 487 nm (ex487) were determined for the YF from both the sucrose gradient (top panel) and thylakoid membranes (bottom panel). The absorbance spectra for each are provided (dashed lines). The absorbance spectrum of the soluble phycobilisome fraction (PBS Abs) is also provided for comparison in the bottom panel. View largeDownload slide Fig. 8 Analysis of total cellular proteins extracted from cultures of C. paradoxa acclimated to LL or HL for 24 h. Proteins from an equivalent number of cells were separated by SDS–PAGE. (A) Phycobilisomes (PBS) were visualized by ZnSO4 staining. (B) CRP was visualized by Western blot analysis with anti-FCP. (C) Changes in D1 were visualized using the anti-D1 antiserum. View largeDownload slide Fig. 8 Analysis of total cellular proteins extracted from cultures of C. paradoxa acclimated to LL or HL for 24 h. Proteins from an equivalent number of cells were separated by SDS–PAGE. (A) Phycobilisomes (PBS) were visualized by ZnSO4 staining. (B) CRP was visualized by Western blot analysis with anti-FCP. (C) Changes in D1 were visualized using the anti-D1 antiserum. Table 1 Pigment content and maximum yield of PSII (FV/FM) in LL- and HL-acclimated C. paradoxa cultures   pg of Chl a cell–1  pg of carotenoid cell–1  FV/FM  Max O2 evolution (fmol cell–1 h–1)  LL  0.202 ± 0.014  0.049 ± 0.003  0.589 ± 0.004  18.6  HL  0.241 ± 0.012  0.076 ± 0.004  0.467 ± 0.028  11.1    pg of Chl a cell–1  pg of carotenoid cell–1  FV/FM  Max O2 evolution (fmol cell–1 h–1)  LL  0.202 ± 0.014  0.049 ± 0.003  0.589 ± 0.004  18.6  HL  0.241 ± 0.012  0.076 ± 0.004  0.467 ± 0.028  11.1  Saturation of oxygen evolution rates occurred at photon flux densities (PFDs) of 100 and 300 µmol photons m–2 s–1 for LL- and HL-acclimated cultures, respectively. View Large Table 2 Pigment content of membranes and free pigments from sucrose density-fractionated membranes of cyanelles isolated from LL- or HL-acclimated C. paradoxa   ng of Chl a  ng of carotenoids  LL      Membranes  65.7 ± 1.1  13.2 ± 0.8  Free pigment  t  2.5 ± 0.4  HL      Membranes  56.6 ± 4.9  13.2 ± 1.0  Free pigment  t  13.4 ± 0.7    ng of Chl a  ng of carotenoids  LL      Membranes  65.7 ± 1.1  13.2 ± 0.8  Free pigment  t  2.5 ± 0.4  HL      Membranes  56.6 ± 4.9  13.2 ± 1.0  Free pigment  t  13.4 ± 0.7  Pigments are expressed as ng µg–1 of protein for membrane fractions and ng µl–1 of sample for free pigment fractions. There were only trace (t) amounts of Chl in the free pigment fractions. 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TI - Isolation of a Novel Carotenoid-rich Protein in Cyanophora paradoxa that is Immunologically Related to the Light-harvesting Complexes of Photosynthetic Eukaryotes
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pci054
DA - 2005-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/isolation-of-a-novel-carotenoid-rich-protein-in-cyanophora-paradoxa-MCim1IYN9L
SP - 416
EP - 424
VL - 46
IS - 3
DP - DeepDyve
ER -