Journal of Oceanology and Limnology   2022, Vol. 40 issue(2): 690-702     PDF       
http://dx.doi.org/10.1007/s00343-021-0451-0
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

CHEN Min, LI Qin, WANG Yuxuan, WANG Jing, ZHANG Kun
The phycocyanin-chlorophyll-protein complexes isolated from Chroomonas placoidea
Journal of Oceanology and Limnology, 40(2): 690-702
http://dx.doi.org/10.1007/s00343-021-0451-0

Article History

Received Dec. 14, 2020
accepted in principle Feb. 27, 2021
accepted for publication Mar. 22, 2021
The phycocyanin-chlorophyll-protein complexes isolated from Chroomonas placoidea
Min CHEN, Qin LI, Yuxuan WANG, Jing WANG, Kun ZHANG     
Institute of Life Science, Yantai University, Yantai 264005, China
Abstract: An active photosystem (PS) Ⅱ particle and two light-harvesting complexes, as well as their sub-complexes that have not been reported previously, were isolated from a cryptophyte Chroomonas placoidea by Triton X-100 sucrose density gradient centrifugation. The fluorescence spectra revealed that there were efficient energy couplings between phycocyanin (PC645) and chlorophyll (Chl) within both zones Ⅲ and Ⅳ of the gradient, which were designated respectively as light-harvesting complex and PSⅡ particles whose size was 15-20 nm according to negative staining in electron microscopy. When the two complexes were further resolved into sub-complexes, the energy coupling was retained in the core PSⅡ complex (named as zone Ⅳ-2 of the sucrose gradient), which contained almost no outer antenna pigment Chl c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the PC645 components appeared in Chl-containing protein complexes were mainly the β subunit with molecular weight of 20 kDa. These results demonstrate that PC645 in this cryptophyte was structurally but preferentially combined with the light-harvesting complex and PSⅡ core. The excitation energy absorbed by PC645 could be directly transferred to Chlα (especially the long wavelength of Chlα) in the PSⅡ reaction center or via the Chl α/c-protein complex. The β subunit corresponded to the terminal fluorescence emission and might play an important role in transmitting energy from PC645 to the Chl-protein complex. The results will help in elucidating the architecture and function of the energy transfer system comprising phycobiliproteins and Chl-protein complexes in cryptophytes.
Keywords: Chroomonas placoidea    phycocyanin-chlorophyll-protein complex    photosystem Ⅱ    lightharvesting complex (LHC)    phycocyanin (PC645)    fluorescence spectra    
1 INTRODUCTION

Oxygen-evolving photosynthetic organisms have evolutionarily conserved reaction centers, which generally contain chlorophyll a (Chl a)-protein complexes and are varied in their types of light-harvesting components, which are Chl a/b-protein complex in green plants, or Chl a/c-protein complex in chromophytic plants and phycobiliproteins (PBPs) in blue and red plants. Cryptophytic algae are chromophytic plants. They are thought to be a unique lineage that emerged from red algae after a secondary endosymbiosis event. Unusually, they possess two sets of co-existed light-harvesting systems in their chloroplast. Some cryptophytes use phycobiliproteins, whose maximal absorption is around 500–650 nm, as their primary light-harvesting complex (LHC). Cryptophytic PBPs are partially inherited from red algae. During the evolution, the phycobilisomes (PBS) in red algae, which are disc-shaped or half disc-shaped multimeric PBPs complexes anchored on the external surface of thylakoid membranes, became reduced and allophycocyanin disappeared. The remaining PBPs, either phycoerythrin (PE) or phycocyanin (PC), depending on the algal species, transferred to the luminal side of the thylakoids (Gantt et al., 1971; Spear-Bernstein and Miller, 1987; Ludwig and Gibbs, 1989), and has evolved into the only type of light-harvesting antenna that located within the thylakoidal lumen found up to now. Although the number of genes encoding PBP subunits differs in different species (Kieselbach et al., 2018), the PBPs in cryptophytes are reported as consisting primarily of α, α′, and β subunits. From crystal structure analysis, PE545 and PC645 were proposed to form a quaternary structure of (αβ) (α′β) heterodimers (Morisset et al., 1984; Wilk et al., 1999; van der Weij-De Wit et al., 2006). The β subunit of cryptophytic PBPs, encoded by the chloroplast genome, has been shown to be inherited from red algae (Douglas and Penny, 1999), while the evolutionary origin of the nuclear-encoded α subunits remain still unknown (Douglas and Penny, 1999; Kieselbach et al., 2018; Greenwold et al., 2019; Tomazic et al., 2020).

Furthermore, as chromophytic algae, cryptophytes contain another type of light-harvesting system in addition to soluble PBPs, namely the insoluble Chl a/c-protein complex. The Chl a/c-protein complex is located predominantly in the stacked regions of thylakoid membranes, and probably mediates the excitation transfer from PE to photosystem (PS) Ⅱ, as determined by steady-state and time-resolved fluorescence measurements in whole cells (Lichtlé et al., 1980, 1987; Ingram and Hiller, 1983; Cheregi et al., 2015). Previous works focused on isolated cryptophytic PBPs, including their crystal structure (Becker et al., 1998; Wilk et al., 1999; Doust et al., 2004; van der Weij-De Wit et al., 2006; Harrop et al., 2014), biological and spectroscopic characteristics (MacColl et al., 1995, 1998; Li and Chen, 2013), and energy transfer (Collini et al., 2010; Novoderezhkin et al., 2010; Arpin et al., 2015). In contrast, the Chl-containing complexes are poorly understood. Generally, 3–6 pigment-protein complexes could be resolved from cryptophytic algae thylakoids. In addition, the results varied from each other under different solubilization and separation conditions (Table 1). Most of them were Chl-protein complexes, only a LHC-PSⅡ fraction (Lichtlé et al., 1987) and a Chl a/c2 were found to combine with cryptophytic PBP (Chen et al., 2007). Actually, the arrangement of cryptophytic PBPs in the thylakoid lumen, as well as the excitation energy transfer pathways between PE or PC to LHC and the reaction centers, which are Chl-containing complexes embedded in the membrane, remain uncertain. It was reported that isolated PE, such as antenna PE545, showed different features of energy transfer compared with those in intact cells (van der Weij-De Wit et al., 2006; Cheregi et al., 2015). It was concluded that spectroscopic experiments on isolated components could not necessarily predict their precise function in intact cells (van der Weij-De Wit et al., 2006). In the present study, active PC-LHC proteins and PC-PSⅡ complexes, as well as their sub-complexes were resolved from a PC645-containing cryptophyte Chroomonas placoidea, and provided a direct view of the composition in sub-complex level, energy transfer and arrangement of PBPs and Chl-protein complexes in this cryptophytic alga.

Table 1 Pigment-protein complexes isolated from cryptophytes
2 MATERIAL AND METHOD 2.1 Isolation and preparation of thylakoid and PC samples

Thylakoids of Chroomonas placoidea were prepared as described previously (Chen et al., 2007) and stored at -70 ℃ in 50-mmol/L Tricine-NaOH buffer (pH 8.0) containing 20% glycerol. Chlorophyll concentration and Chl a/c ratio of thylakoids and isolated zones were determined following the procedure of Jeffrey and Humphrey (1975). The blue supernatant containing PC was treated with 70% ammonium sulfate overnight and centrifuged at 36 000×g for 10 min to deplete thylakoids and Chl-containing components. The supernatant was then precipitated with 100% ammonium sulfate. The PC sediment was collected by centrifuging at 2 000×g for 20 min, dialyzed against 0.5-mol/L phosphate buffer (pH 7.5) overnight, and stored at 4 ℃ in darkness.

2.2 Sucrose gradient centrifugation of pigment–protein complexes

Before sucrose gradient centrifugation, stored thylakoids were thawed and washed twice with 7.5-mmol/L EDTA (pH 8.0) containing 50-mmol/L sorbitol and centrifuged at 36 000×g for 10 min. The thylakoid sediments were re-suspended in ultrapure water at 0.8-mg/mL Chl and adjusted to pH 8.0 using (CH3)4NOH. Then, Triton X-100 was added to a final concentration of 0.7%. The mixture was stirred at 4 ℃ for 30 min, then centrifuged at 30 000×g for 10 min. Aliquots (0.5–1.0 mL) of the supernatant were immediately loaded onto a 5-mL SW-41 centrifuge tube containing a discontinuous sucrose gradient comprising 1.5 mol/L (1.0 mL), 0.8 mol/L (0.5 mL), 0.6 mol/L (0.5 mL), 0.5 mol/L (1 mL), 0.4 mol/L (1 mL), and 0.3 mol/L (0.5 mL), each containing 0.02% Triton X-100. The gradients were centrifuged in a Hitachi-55P-72 SW-41 centrifuge at 220 000×g for 14–16 h.

2.3 Secondary sucrose gradient centrifugation of pigment–protein sub-complex

Zones containing pigments were collected, pooled and diluted in 50-mmol/L Tricine- NaOH buffer (pH 8.0), then centrifuged and concentrated at 38 000×g for 7 h in 10-kDa ultra-filtration tubes to deplete sucrose. The concentrates were adjusted to 0.5-mg/ mL Chl with (CH3)4NOH (pH 8.0), and 20% Triton X-100 was added to a final concentration of 0.5%. The mixtures were incubated at 4 ℃ for 10 min in darkness, then subjected to a secondary step of centrifugation in 12-mL tubes containing discontinuous sucrose gradients comprising 1.5 mol/L (2.5 mL), 1.0 mol/L (1.0 mL), 0.8 mol/L (1.0 mL), 0.6 mol/L (1.0 mL), 0.5 mol/L (2.0 mL), 0.4 mol/L (1.0 mL), and 0.3 mol/L (1.0 mL) under the same centrifugal conditions described above.

2.4 Measurement of photo-oxidative activity

For assay of PSⅠ activity, dichlorophenol indophenol (DCPIPH2) oxidation was performed on fractions collected from sucrose gradients as described by the Teaching and Research Group of Plant Physiology, Department of Biology, East China Normal University (1980). Measurement of PSⅡ activity was carried out as described by Samuelsson and Prezélin (1985).

2.5 Negative staining

Portions of 5 μL of zone Ⅳ from the sucrose gradient were applied at 0.03 mg/mL or 1.5 mg/mL to the surface of hydro-treated and carbon-coated copper grids then stained for 1 min. The grids were washed with ddH2O after removing the drops, and then dyed with 5-μL 2% uranyl acetate for 1 min. Samples were detected at 200 kV by transmission electron microscopy (Talos F200C), and images were recorded by camera (Ceta) with a magnification of 73 000×g.

2.6 Absorption and fluorescence spectra

Thylakoids and zones collected from sucrose gradients were diluted with 50-mmol/L Tricine-NaOH (pH 8.0). Absorption spectra were recorded on an UV-3400 spectrophotometer, and fluorescence spectra at room temperature or 77 K (-196 ℃) were recorded with a Carian Cary Eclipse fluorimeter. Glycerol (50%) was added to samples before low temperature spectrum determinations.

2.7 Polypeptide analysis by SDS-PAGE

Thylakoids, PC samples, as well as pigment-protein complexes and sub-complexes isolated by sucrose gradient centrifugation were precipitated with 20% trichloroacetic acid in microcentrifuge tubes, and centrifuged at 25 000×g for 10 min. The sediments (except PC samples) were washed twice with 80% acetone/alcohol to deplete chlorophylls and carotenoids. All samples were incubated in sample treating buffer (0.1-mol/L Tris-HCl buffer, pH 6.8, containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.005% bromophenol blue) for 3 h at 37 ℃. Thylakoids were centrifuged at 30 000×g for 10 min before SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Electrophoresis was carried out according to Laemmli (1970), and the gels were fixed in 0.2-mol/L iminazole for 10min and stained with 0.2-mol/L ZnSO4, which enhances the fluorescent intensity of the PC subunit. Gels were observed and photographed under ultra-violet light in a UVP (Ultra-Violet Products)-Biospectrum imaging system, washed with 7% (v/v) acetic acid to deplete ZnSO4, and photographed under natural light after restaining by Coomassie brilliant blue G-25.

3 RESULT 3.1 Thylakoids of C. placoidea

The absorption spectrum of well-washed C. placoidea thylakoids showed absorption maxima corresponding to Chl a (438 and 677 nm), Chl c (464 nm for the blue region; the red region absorption was covered by PC645), carotenoids (490 nm), and PC645 (580- and 645-nm typical absorption peaks and 625-nm shoulder) (Fig. 1a). Low-temperature fluorescence emission spectra showed that exciting Chl a at 436 nm and Chl c at 460 nm led to PSⅡ-specific emission peaks with maximum at 692 nm coupled with a shoulder at 685 nm (Fig. 1b). The main contributor to the two maxima is most likely the inner antenna of PSⅡ, as in higher plants and blue algae. In the emission spectrum with excitation at 580 nm, the 685-nm peak disappeared and the 692-nm peak diminished, while the maximum emission migrated to 703 nm. The 662-nm individual fluorescent emission indicated the presence of PC645, and the 716-nm shoulder represented the activity of PSI. This result was unusual, and suggested that the PC645 might energetically connect to PSI in the thylakoids of C. placoidea, unlike the PE545 in cells of Guillardia theta, primarily connected to PSⅡ (Kieselbach et al., 2018).

Fig.1 The absorption spectra of thylakoid and PC645 at room temperature (a) and fluorescence emission spectra at 77 K (b)
3.2 Isolation of pigment-protein complexes

Centrifugation of Triton X-100-solubilized thylakoids (Chl a/c ratio was 4.04) on discontinuous sucrose density gradients gave five distinct fractions, named as zones Ⅰ–Ⅴ from top to bottom (Fig. 2b). Zone Ⅰ, located at the top of the tube, was orange and contained no proteins in SDS-PAGE (data not shown). It was characterized as a free pigment band.

Fig.2 Triton X-100 sucrose gradient centrifugation of PC645 (a) and thylakoids (b)

The dark green fraction zone Ⅱ was at 0.3–0.4-mol/L sucrose gradient. It contained nearly 50% of the total chlorophyll, including Chl c (absorption at 460–465 and 628 nm) and Chl a (436 and 672 nm) (Chl a/c ratio=2.91), as well as carotenoids (490–495 nm) (Fig. 3a), and showed neither PSI nor PSⅡ oxidative activity. The excitation spectrum of zone Ⅱ showed the excitation peaks of Chl a (437 and 672 nm) (Fig. 3b). However, Chl c contributed less to the 700-nm fluorescence emission, compared with zones Ⅲ and Ⅳ. The 464–466-nm fluorescence in the blue region, associated with Chl c, was weak (Fig. 3b). The maximum emission of zone Ⅱ was at 679–680 nm when the fraction was excited at 436, 460, or 580 nm (Fig. 4a). These results suggest that zone Ⅱ might be a light-harvesting Chl a/c-protein complex that maintains a certain extent of energy transfer among the pigments.

Fig.3 The absorption (a) and fluorescence excitation spectra (b) of pigment-protein complexes isolated by gradient centrifugation at room temperature (Em=700 nm)
Fig.4 Fluorescence emission spectra of zones isolated by sucrose gradient centrifugation determined at room temperature a. Zone Ⅱ; b. Zone Ⅲ; c. Zone Ⅳ.

The yellow-green band zone Ⅲ at 0.5-mol/L sucrose had no photo-chemical activity. It had two small absorption peaks at 587 and 642 nm that probably corresponded to PC645 in addition to major peaks for Chl a (438 and 672 nm) and relative higher amounts of Chl c (464 nm) and carotenoids (495 nm) compared to the other isolated zones (Fig. 3a) (Chl a/c ratio=1.47). The fluorescence emission spectra of zone Ⅲ were unusual. The dominant fluorescence emission excited by either 436- or 460-nm excitation light was at 680–681 nm, similar to that for zone Ⅱ (Fig. 4b). However, the emission spectrum excited by 580-nm light showed a peak at 675 nm (684 nm at 77 K, Fig. 5a) coupled with a shoulder around 661 nm (minimized shoulder at 660–665 nm at 77 K). The excitation spectra at 700 nm also showed a fluorescence contribution of PC at 589 nm and 644 nm (Fig. 3b). Accordingly, zone Ⅲ might be a PC-Chl a/c-protein light-harvesting complex, although some free PC components might co-migrate with it (Fig. 2a).

Fig.5 Fluorescence emission spectra determined at 77 K

Zone Ⅳ was a green band located at 0.6-mol/L sucrose gradient. The absorption spectra showed the dominant absorptions were long wavelength of Chl a (438 nm and 676–678 nm) and carotenoids (495–497 nm) (Fig. 3a). A small amount of Chl c (464 nm) and trace of PC component also appeared (Chl a/c ratio=6.62); the latter absorbed mainly around 645 nm and made the Chl c absorption band in the red region migrate from 625–630 to 632–635 nm. When excited by 580-nm light, zone Ⅳ showed a fluorescent emission coupling of 662–665 nm from PC645 and a peak at 685 nm from Chl a at room temperature (Fig. 4c), and 663–668 nm coupled with 680–682 nm at 77 K (Fig. 5b), indicating energy transfer from PC to Chl a in this complex. This result was confirmed by the excitation spectra (Fig. 3b): the excitation peaks at 589 and 640–644 nm were attributed to PC. Furthermore, zone Ⅳ showed obvious PSⅡ photo-oxidative activity. These features allowed us to designate zone Ⅳ as an active PC-Chl a/c-protein PSⅡ complex. This particle was round in shape with a diameter size about 15–20 nm, according to negative staining and transmission electron microscopy measurement (Fig. 6a & b).

Fig.6 Negative staining of zone Ⅳ observed by cryogenic transmission electron microscopy The concentration of samples was 0.03 mg/mL (a) and 1.5 mg/mL (b).

Zone V (yellow-green), which appeared at 1.0 mol/L in the sucrose gradient, showed P700 oxidative activity was therefore characterized as an active PSI particle. The results of isolated zones after the first steps of sucrose gradient centrifugation are summarized in Table 2.

Table 2 The pigment-protein complexes isolated by the first step of sucrose gradient centrifugation
3.3 Sub-complex isolated by secondary Triton X-100 sucrose gradient centrifugation

Light-harvesting complexes (zones Ⅱ, Ⅲ) and PSⅡ particle (zone Ⅳ) were pooled and subjected to secondary Triton X-100 sucrose gradient centrifugation. Zone Ⅱ-1 migrated at the same position (Fig. 7a) and showed similar absorbance and fluorescence features to zone Ⅱ (results not shown). Zone Ⅲ created a new yellow-green band at 0.3 mol/L in the gradient, named as zone Ⅲ-1, while zone Ⅲ-2 appeared at a similar gradient position as zone Ⅲ in the first centrifugation (Fig. 7b). Zone Ⅲ-2 contained an even higher amount of Chl c (460 nm) and carotenoids (495 nm) than did zone Ⅲ (Fig. 8a), while zone Ⅲ-1 was lower. The 642-nm fluorescence emission peak in zone Ⅲ-1 produced by Chl c indicated the partial interruption of energy transfer from Chl c to Chl a (Fig. 9a). The PC component (585 and 640 nm) remained in zone Ⅲ-2 (Fig. 8a), which contributed 665-nm fluorescence emission shoulder except 680-nm fluorescence of Chl a under excitation by 580-nm light, and 589- and 642-nm fluorescence under excitation at 700-nm emission light (Fig. 9b). This result was confirmed by low temperature fluorescence emission spectra (Fig. 10a).

Fig.7 Secondary sucrose gradient centrifugation of zone Ⅱ (a), zone Ⅲ (b), and zone Ⅳ (c) treated with 0.5% Triton X-100 before centrifugation
Fig.8 Absorption spectra of zones isolated by secondary sucrose gradient centrifugation after further treatment with 0.5% Triton X-100
Fig.9 Room temperature fluorescence spectra of sub-complexes isolated by secondary sucrose gradient centrifugation a. Zone Ⅲ-1; b. Zone Ⅲ-2; c. Zone Ⅳ-1; d. Zone Ⅳ-2.
Fig.10 Fluorescence emission spectra of sub-complexes determined at 77 K a. Zone Ⅲ-2; b. Zone Ⅳ-2.

Zone Ⅳ also created two bands, named zone Ⅳ-1, located at 0.3 mol/L in the gradient, and zone Ⅳ-2 at 0.6 mol/L (Fig. 7c). Zone Ⅳ-1, containing a small amount of light-harvesting complex stripped from PSⅡ core particle (zone Ⅳ), showed a small amount of Chl c (464 and 625 nm) and carotenoids (494 nm) (Fig. 8b). The fluorescence contribution of Chl c and PC were not obvious to 700 nm (Fig. 9c). The green band zone Ⅳ-2, containing mainly Chl a (438 and 677 nm), small amount of carotenoids (494 nm) and only trace of Chl c (464 nm), showed obvious PSⅡ photo-oxidative activity, and was characterized as a PSⅡ core complex. Significantly, zone Ⅳ-2 retained a 660–664-nm and 685–688-nm coupling of fluorescence emission under 580-nm excitation at room temperature (Fig. 9d) and 644 nm with 673 nm coupling peak at 77 K (Fig. 10b). Therefore, zone Ⅳ-2 appears to be a core complex of PSⅡ still tightly associated with PC components; this finding has not been reported before in cryptophytic algae.

3.4 Fluorescence dynamics during storage

Light-harvesting sub-complex zone Ⅲ-2 and PSⅡ complex zone Ⅳ were collected and stored at room temperature (15–25 ℃) for 7 days. The fluorescence emission spectra when excited with 580-nm light showed that the 680-nm or 685-nm peak emitted by Chl a became lower during storage, while the 660-nm fluorescence of PC became higher (Fig. 11a & b). This result confirmed that the PC components were combined non-covalently with chlorophyll-protein complexes and were able to transfer excitation energy efficiently to Chl a in both light-harvesting and PSⅡ core complexes. Storage can cause the decomposition of PC from Chl-containing protein complexes, as well as the interruption of energy transfer between PC645 bilins and Chl a.

Fig.11 Effect of storage on fluorescence of zone Ⅲ-2 (a) and sub-complex zone Ⅳ (b) at room temperature (Ex=580 nm)
3.5 Polypeptide analysis by SDS-PAGE

Zones enriched in PSⅡ and light-harvesting complexes isolated by two steps of sucrose gradient centrifugation and PC samples were subjected to SDS-PAGE. The polypeptide patterns of PC samples revealed the presence of three main polypeptides of relative molecular masses (Mr) approximately 20 and 10 kDa, which were reported as the β and α subunits of PC645 (Lichtlé et al., 1987), and an 8-kDa subunit named as α′ (Fig. 12a & b). All three subunits showed pink fluorescence under ultra-violet light after staining by ZnSO4 (Fig. 12b). Furthermore, some peptides at the 30–40-kDa region also showed pink fluorescence and were considered as αβ and ββ aggregates (Fig. 12b).

Fig.12 Peptide profiles of chlorophyll-protein complexes and sub-complexes stained with Coomassie brilliant blue (a) and ZnSO4 under UV light (b)

The small LHC zone Ⅱ contained mainly peptides of 18–24 kDa (Fig. 12) that were considered typical LHC proteins as previously identified by the Western blotting (Janssen and Rhiel, 2008). Zone Ⅲ, a larger Mr light-harvesting fraction, comprised a multitude of peptides ranging from under 5 kDa up to 65 kDa, dominated by peptides of 18, 24, 25, 28, 34, and 45 kDa. The sub-complex zone Ⅲ-2 showed a similar composition to zone Ⅲ, but zone Ⅲ-1 was found to consist of peptides of 40–65 kDa, as was sub-complex zone Ⅳ-1. Zone Ⅳ, which was characterized as PSⅡ particles, consisted of three prominent groups of peptides of around 18–28, 30–36, and 40–60 kDa, although some peptides with higher Mr were also detected. Zone Ⅳ-2 contained mostly peptides of 30–66 kDa, which were designated as PSⅡ peptides (Ingram and Hiller, 1983; Lichtlé et al., 1987; Bathke et al., 1999; Janssen and Rhiel, 2008), and two smaller Mr peptides of 16 and 20 kDa. The 18–28-kDa LHC proteins of zone Ⅳ were diminished in zone Ⅳ-2 because of the further resolution and separation. Zone Ⅳ-2 was characterized as a core PSⅡ complex with a small amount of associated LHC remaining. Significantly, zones Ⅲ, Ⅳ, Ⅲ-2, and Ⅳ-2 also contained an extra peptide of 20 kDa, and showed the same migration position and pink fluorescence under ultra-violet as the β subunit in PC (Fig. 12b). Sometimes the 20-kDa subunits aggregated as a ββ complex and showed another less intense fluorescent band at around 40 kDa (Fig. 12). In contrast, the α subunits were not noticeable in these Chl-containing complexes, when stained with either ZnSO4 or silver (data not shown).

4 DISCUSSION

Cryptophytes are unique organisms that contain both PBPs and chlorophyll-protein complexes as their light-harvesting structures. Investigations on the photosystems of cryptophytes have focused on isolated PBPs rather than on Chl-containing proteins. Fractions enriched in PSI, PSⅡ, and Chl a/c2-protein complexes have been reported following either gel electrophoresis or sucrose density centrifugation (Ingram and Hiller, 1983; Lichtlé et al., 1987; Janssen and Rhiel, 2008). However, the potential sub-complexes of these fractions have not been reported.

The latest model of cryptophytic algal photosystems suggested that the cryptophytic PBPs pack densely and freely inside the lumen as individual heterodimers displaying no relation to each other or to the thylakoid membrane (Doust et al., 2004; Mirkovic et al., 2017). An active PE-PSⅡ complex from Cryptomonas rufescens and a novel PC-Chl a/c2-protein complex in C. placoidea were reported by Lichtlé et al. (1987) and Chen et al. (2007), respectively. Here we provide evidence for two kinds of PC-PSⅡ complexes as well as two PC-LHC complexes. Fluorescent spectra and a dynamic study during storage confirmed that the PC645 was combined non-covalently with chlorophyll-protein complexes in these isolated complexes. Efficient energy transfer was observed from PC645 to Chl a in both PSⅡ particles and LHCs. These results demonstrate that PC645 in C. placoidea is structurally but not preferentially combined with a LHC and the PSⅡ reaction center. The excitation energy absorbed by PC645 can be directly transferred to Chl a in the PSⅡ reaction center or via a Chl a/c-protein complex. The present work provides a series of PC-chlorophyll-protein complexes which should serve as useful materials for further investigation of structural architecture, energy transfer, and the molecular arrangement of photosynthetic systems in cryptophytic algae.

From previous peptide analysis and western blotting (Janssen and Rhiel, 2008), the proteins of 18–22 kDa, which we found as the dominant peptides in zones Ⅱ, Ⅲ, and Ⅲ-2, are considered typical LHC proteins of cryptophytic algae. The peptides of 30–34 kDa enriched in zone Ⅳ and sub-complex zone Ⅳ-2, which are characterized as PSⅡ particles and reaction cores, respectively, are considered the core peptides D1/D2 of PSⅡ. In the previous report, the proteins at 45–55 kDa were designated as inner antenna peptides of PSⅡ; if the PSⅡ in cryptophytes has a similar inner antenna structure to that in higher plants and green algae, it has not yet been well defined. Accordingly, in the present work, zone Ⅱ (containing 18–22-kDa peptides) was considered as a low Mr LHC associated peripherally with the PSⅡ core. Zone Ⅲ (an intact LHC with some PSⅡ components still attached), was divided into two parts (Fig. 7): the inner antenna zone Ⅲ-1 containing inner peptides and less Chl c, and zone Ⅲ-2 containing unusually high amounts of auxiliary pigments Chl c and PC. Zone Ⅲ-2 was proposed as a large-size LHC located at the outer part of PSⅡ. Zone Ⅳ is a larger PSⅡ particle with a diameter of about 15–20 nm, similar in size to the PSⅡ dimer of blue algae (Zhao et al., 2020), but smaller than the PSⅡ-LHCⅡ particle of spinach that was reported as 26 nm × 14 nm ×11 nm (Wei et al., 2016) because of the attachment of two tightly associated LHCⅡ trimers along the long axis. Therefore, zone Ⅳ was considered as a PSⅡ particle surrounded by a certain amount of antenna LHC. Zone Ⅳ-1, derived directly from zone Ⅳ that contained active PSⅡ particles, was considered as the inner antenna of PSⅡ that might be located closer to the reaction core complex than are the proteins of zone Ⅲ-1. The other part of zone Ⅳ, named zone Ⅳ-2, was a typical core complex of PSⅡ with traces of LHC peptides and PC subunits still attached. The proposed relationship of these isolated complexes and sub-complexes is summarized in Fig. 13.

Fig.13 Sketch diagram of LHC and PSII complex in C. placoidea

Each PC645 heterodimer contained three different types of bilins (Wedemayer et al., 1996; Collini et al., 2010; Overkamp et al., 2014): one 15, 16-dihydrobiliverdin (DBV) and two phycocyaninbilins (PCB) in each β subunit, and one mesobiliverdin (MBV) in each α subunit. MBV exhibits a strong red absorption at 697 nm and had been considered as the terminal emitter bilin in the PC645 heterodimer (Doust et al., 2006). But ultrafast spectral analysis elucidated that the shoulder at 625–633 nm and the red most peak at 640–645 nm were assigned to four PCBs. The PCB β82 was assumed to be the final emitter and responsible for the characteristic fluorescence emission of PC645 at 660–665 nm (Mirkovic et al., 2007, 2017; Marin et al., 2011). In our case, two small fluorescent α subunits with Mr of 8 kDa and 10 kDa were detected in addition to the 20-kDa β subunit in our purified PC645 sample as described in previous reports. However, in our isolated PC-Chl-protein complexes, only β subunits rather than α subunits could be detected. These Chl-containing complexes were obtained from well-washed thylakoids, a process that might cause the loss of small α subunits. When the excitation wavelength was 580 nm, these complexes exhibited the 670–685-nm fluorescence emitted by Chl a, coupled with 660–665-nm emission from the final emitting bilins of PC645. Therefore, the demonstration of 20-kDa β subunits in these PC-Chl-protein complexes provides direct evidence supporting the view that the β subunit corresponds to the terminal emission of PC645. These results also imply that PC645 might attach to the insoluble thylakoid components with a specific form and angle. The β subunit might play an important role in the combination of PC645 with Chl-containing complexes, although the α subunits also contribute importantly to interactions in the quaternary structure of PC645 and energy transfer between PC645 and chlorophylls (van der Weij-De Wit et al., 2006). Further investigation on the precise structure and energy transfer of these novel PC-Chl-protein complexes will help in elucidating the characteristics and function of these subunits in the structural relationship between PBPs and Chl-protein complexes in cryptophytes.

5 DATA AVAILABILITY STATEMENT

The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.

References
Arpin P C, Turner D B, McClure S D, Jumper C C, Mirkovic T, Challa J R, Lee J, Teng C Y, Green B R, Wilk K E, Curmi P M G, Hoef-Emden K, McCamant D W, Scholes G D. 2015. Spectroscopic studies of cryptophyte light harvesting proteins: vibrations and coherent oscillations. The Journal of Physical Chemistry B, 119(31): 10 025-10 034. DOI:10.1021/acs.jpcb.5b04704
Bathke L, Rhiel E, Krumbein W E, Marquardt J. 1999. Biochemical and Immunochemical investigations on the light-harvesting system of the cryptophyte Rhodomonas sp.: evidence for a photosystem I specific antenna. Plant Biology, 1(5): 516-523. DOI:10.1111/j.1438-8677.1999.tb00777.x
Becker M, Stubbs M T, Huber R. 1998. Crystallization of phycoerythrin 545 of Rhodomonas lens using detergents and unusual additives. Protein Science, 7(3): 580-586. DOI:10.1002/pro.5560070306
Chen M, Li S H, Sun L. 2007. A novel phycocyamn-Chla/c2-protein complex isolated from chloroplasts of Chroomonas placoidea. Chinese Chemical Letters, 18(11): 1 374-1 378. DOI:10.1016/j.cclet.2007.09.025
Cheregi O, Kotabová E, Prášil O, Schröder W P, Kaňa R, Funk C. 2015. Presence of state transitions in the cryptophyte alga Guillardia theta. Journal of Experimental Botany, 66(20): 6 461-6 470. DOI:10.1093/jxb/erv362
Collini E, Wong C Y, Wilk K E, Curmi P M G, Brumer P, Scholes G D. 2010. Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature. Nature, 463(7281): 644-647. DOI:10.1038/nature08811
Douglas S E, Penny S L. 1999. The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae. Journal of Molecular Evolution, 48(2): 236-244. DOI:10.1007/pl00006462
Doust A B, Marai C N J, Harrop S J, Wilk K E, Curmi P M G, Scholes G D. 2004. Developing a structure-function model for the cryptophyte phycoerythrin 545 using ultrahigh resolution crystallography and ultrafast laser spectroscopy. Journal of Molecular Biology, 344(1): 135-153. DOI:10.1016/j.jmb.2004.09.044
Doust A B, Wilk K E, Curmi P M G, Scholes G D. 2006. The photophysics of cryptophyte light-harvesting. Journal of Photochemistry and Photobiology A: Chemistry, 184(1-2): 1-17. DOI:10.1016/jophotochem.2006.06.006
Gantt E, Edwards M R, Provasoli L. 1971. Chloroplast structure of the Cryptophyceae: evidence for phycobiliproteins within intrathylakoidal spaces. Journal of Cell Biology, 48(2): 280-290. DOI:10.1083/jcb.48.2.280
Greenwold M J, Cunningham B R, Lachenmyer E M, Pullman J M, Richardson T L, Dudycha J L. 2019. Diversification of light capture ability was accompanied by the evolution of phycobiliproteins in cryptophyte algae. Proceedings of the Royal Society B: Biological Sciences, 286(1902): 20190655. DOI:10.1098/rspb.2019.0655
Harrop S J, Wilk K E, Dinshaw R, Collini E, Mirkovic T, Teng C Y, Oblinsky D G, Green B R, Hoef-Emden K, Hiller R G, Scholes G D, Curmi P M G. 2014. Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins. Proceedings of the National Academy of Sciences of the United States of America, 111(26): E2 666-E2 675. DOI:10.1073/pnas.1402538111
Ingram K, Hiller R G. 1983. Isolation and characterization of a major chlorophyll ac2 light-harvesting protein from a Chroomonas species (Cryptophyceae). Biochimica et Biophysica Acta (BBA) — Bioenergetics, 722(2): 310-319. DOI:10.1016/0005-2728(83)90078-6
Janssen J, Rhiel E. 2008. Evidence of monomeric photosystem I complexes and phosphorylation of chlorophyll a/c-binding polypeptides in Chroomonas sp. strain LT (Cryptophyceae). International Microbiology, the Official Journal of the Spanish Society for Microbiology, 11(3): 171-178.
Jeffrey S W, Humphrey G F. 1975. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochemie und Physiologie der Pflanzen, 167(2): 191-194. DOI:10.1016/S0015-3796(17)30778-3
Kieselbach T, Cheregi O, Green B R, Funk C. 2018. Proteomic analysis of the phycobiliprotein antenna of the cryptophyte alga Guillardia theta cultured under different light intensities. Photosynthesis Research, 135(1): 149-163. DOI:10.1007/s11120-017-0400-0
Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685. DOI:10.1038/227680a0
Li W J, Chen M. 2013. Structural and functional stability of phycocyanin from Chroomonas placoidea. Marine Sciences, 37(7): 33-40. (in Chinese with English abstract)
Lichtlé C, Duval J C, Lemoine Y. 1987. Comparative biochemical, functional and ultrastructural studies of photosystem particles from a Cryptophycea: cryptomonas rufescens; isolation of an active phycoerythrin particle. Biochimica et Biophysica Acta (BBA) — Bioenergetics, 894(1): 76-90. DOI:10.1016/0005-2728(87)90214-3
Lichtlé C, Jupin H, Duval J C. 1980. Energy transfers from Photosystem I to Photosystem I in Cryptomonas rufescens (Cryptophyceae). Biochimica et Biophysica Acta (BBA) — Bioenergetics, 591(1): 104-112. DOI:10.1016/0005-2728(80)90224-8
Ludwig M, Gibbs S P. 1989. Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens. Journal of Cell Biology, 108(3): 875-884. DOI:10.1083/jcb.108.3.875
MacColl R, Malak H, Cipollo J, Label B, Ricci G, MacColl D, Eisele L E. 1995. Studies on the dissociation of cryptomonad biliproteins. Journal of Biological Chemistry, 270(46): 27 555-27 561. DOI:10.1074/jbc.270.46.27555
MacColl R, Malak H, Gryczynski I, Eisele L E, Mizejewski G J, Franklin E, Sheikh H, Montellese D, Hopkins S, MacColl L C. 1998. Phycoerythrin 545: monomers, energy migration, bilin topography, and monomer/dimer equilibrium. Biochemistry, 37(1): 417-423. DOI:10.1021/bi971453s
Marin A, Doust A B, Scholes G D, Wilk K E, Curmi P M G, van Stokkum I H M, van Grondelle R. 2011. Flow of excitation energy in the cryptophyte light-harvesting antenna phycocyanin 645. Biophysical Journal, 101(4): 1 004-1 013. DOI:10.1016/j.bpj.2011.07.012
Mirkovic T, Doust A B, Kim J, Wilk K E, Curutchet C, Mennucci B, Cammi R, Curmi P M G, Scholes G D. 2007. Ultrafast light harvesting dynamics in the cryptophyte phycocyanin 645. Photochemical & Photobiological Sciences: Official Journal of the European Photochemistry Association and the European Society for Photobiology, 6(9): 964-975. DOI:10.1039/b704962e
Mirkovic T, Ostroumov E E, Anna J M, van Grondelle R, Govindjee, Scholes G D. 2017. Light absorption and energy transfer in the antenna complexes of photosynthetic organisms. Chemical Reviews, 117(2): 249-293. DOI:10.1021/acs.chemrev.6b00002
Morisset W, Wehrmeyer W, Schirmer T, Bode W. 1984. Crystallization and preliminary x-ray diffraction data of the cryptomonad biliprotein phycocyanin-645 from a Chroomonas spec. Archives of Microbiology, 140(2): 202-205. DOI:10.1007/BF00454927
Novoderezhkin V I, Doust A B, Curutchet C, Scholes G D. 2010. Excitation dynamics in phycoerythrin 545: modeling of steady-state spectra and transient absorption with modified Redfield theory. Biophysical Journal, 99(2): 344-352. DOI:10.1016/j.bpj.2010.04.039
Overkamp K E, Langklotz S, Aras M, Helling S, Marcus K, Bandow J E, Hoef-Emden K, Frankenberg-Dinkel N. 2014. Chromophore composition of the phycobiliprotein Cr-PC577 from the cryptophyte Hemiselmis pacifica. Photosynthesis Research, 122(3): 293-304. DOI:10.1007/s11120-014-0029-1
Samuelsson G, Prezélin B B. 1985. Photosynthetic electron transport in cell-free extracts of diverse phytoplankton. Journal of Phycology, 21: 453-457. DOI:10.1111/j.0022-3646.1985.00453.x
Spear-Bernstein L, Miller K R. 1987. Immunogold localization of the phycobiliprotein of a cryptophyte alga to the intrathylakoidal space. In: Biggins J ed. Progress in Photosynthesis Research. Martinus Nijhoff Publishing, the Netherlands. p. 309–312.
Teaching and Research Group of Plant Physiology, Department of Biology, East China Normal University. 1980. Guidance of Plant Physiological Experiment. People's Education Publication, Beijing. p. 116–117. (in Chinese)
Tomazic N, Overkamp K E, Aras M, Pierik A J, Hofmann E, Frankenberg-Dinkel N. 2020. Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity. BioRxiv. DOI:10.1101/2020.03.31.018853
van der Weij-De Wit C D, Doust A B, van Stokkum I H M, Dekker J P, Wilk K E, Curmi P M G, Scholes G D, van Grondelle R. 2006. How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem Ⅱ in Cryptophyte Rhodomonas CS24 Cells. The Journal of Physical Chemistry B, 110(49): 25 066-25 073. DOI:10.1021/jp061546w
Wedemayer G J, Kidd D G, Glazer A N. 1996. Cryptomonad biliproteins: bilin types and locations. Photosynthesis Research, 48(1): 163-170. DOI:10.1007/BF00041006
Wei X P, Su X D, Cao P, Liu X Y, Chang W R, Zhang X Z, Liu Z F. 2016. Structure of spinach photosystem Ⅱ-LHCⅡ supercomplex at 3.2 Å resolution. Nature, 534(7605): 69-74. DOI:10.1038/nature18020
Wilk K E, Harrop S J, Jankova L, Edler D, Keenan G, Sharples F, Hiller R G, Curmi P M G. 1999. Evolution of a light-harvesting protein by addition of new subunits and rearrangement of conserved elements: crystal structure of a Cryptophyte phycoerythrin at 1.63-Å resolution. Proceedings of the National Academy of Sciences of The United States of America, 96(16): 8 901-8 906. DOI:10.1073/pnas.96.16.8901
Zhao L S, Huokko T, Wilson S, Simpson D M, Wang Q, Ruban A V, Mullineaux C W, Zhang Y Z X, Liu L N. 2020. Structural variability, coordination and adaptation of a native photosynthetic machinery. Nature Plants, 6(7): 869-882. DOI:10.1038/s41477-020-0694-3