Thylakoid membranes of the thermophilic cyanobacterium Phormidium laminosum have been fractionated into photosystem II and photosystem I particles. These fractions have been characterized by their partial electron transport activities, and biochemical and spectral properties. Exoplasmic fracture face and protoplasmic fracture face particles in the unfractionated thylakoid membranes were shown to correspond in size to particles in freeze-fractured photosystem II and photosystem I fractions, respectively. Differences between the histograms of the thylakoid membrane protoplasmic fracture face particles and the isolated photosystem I particles suggest that in addition to photosystem I complexes some of the particles on the thylakoid protoplasmic fracture face may be related to cytochrome b/f complexes, the hydrophobic component of the coupling factor, or respiratory complexes.

The characterization of particles in thylakoid membranes revealed by freeze-fracture electron microscopy has been the subject of several studies, which have indicated a correlation between intramembranous particles and photosynthetic processes. Arntzen, Dilley & Crane (1969) demonstrated that chloroplast membrane fractions enriched in photosystem II (PS II) were also enriched in large exoplasmic fracture face (E-face) particles, while fractions containing mostly photosystem I (PS In red algae and cyanobacteria, the basic photosynthetic apparatus is analogous to that in higher plants and algae containing chlorophyll b; water is the electron donor and oxygen is evolved, there are two pigment systems with reaction centres based on chlorophyll a, and the electron transport components are comparable. The main difference between higher plant and red algal/cyanobacterial photosynthesis is in their respective light-harvesting apparatus; the chlorophyll a/b pigment protein complex is found in the former and phycobiliproteins in the latter. The phycobiliproteins are organized into highly ordered structures, phycobilisomes (PBsomes), which are attached to the stromal, or outer, surface of the thylakoid membrane.

Consistent with their function as light harvesters, PBsomes are energetically coupled to the chlorophyll proteins and reaction centres in the membrane. Studies have indicated that light energy absorbed by PBsomes is transferred preferentially to PS II (Ley & Butler, 1976; Wang, Stevens & Myers, 1977). Diner (1979) showed that in the case of the red alga, Cyanidium caldarium, as few as half of the PBsomes were functionally connected with PS II centres. More recently, on the other hand, it has been found that the PBsomes of the cyanobacterium, Anacystis nidulans, are functionally (and presumably structurally) connected to two PS II centres, resulting in a competition for excitation energy (Mandori & Melis, 1985).

Freeze-fracture studies of thylakoids from several cyanobacterial and red algal species have revealed, as is the case in higher plants, two basic types of intra-membranous particles associated with thylakoid membranes: somewhat larger E-face particles and smaller, more densely packed P-face particles (Wollman, 1979; Golecki, 1979; Giddings & Staehelin, 1979; Cox, Benson & Dwarte, 1981; Giddings, Wassman & Staehelin, 1983). On the whole, size differences between Eface and P-face particles are greater in higher plants than in PBsome-containing organisms, probably due to the chlorophyll a/b protein complex component of higher plant E-face particles. In the red alga, Spermothamnion turneri, the particles on both fracture faces are similar in size, but the spread of P-face particle sizes is greater than that for the E-face, which suggested that several categories of similarly sized particles might be present in the P-face (Staehelin, Giddings, Badami & Krzymowski, 1978). An additional feature of the intramembranous particles of some of the organisms studied is that ordered rows of E-face particles have been observed (Neushul, 1971; Lefort-Tran, Cohen-Bazire & Pouphile, 1973; Lichtle & Thomas, 1976; Wollman, 1979; Golecki, 1979; Giddings & Staehelin, 1979; Giddings et al. 1983). Lichtle & Thomas (1976) and Giddings et al. (1983) have correlated the spacing of PBsome rows to E-face particle rows, suggesting that the PBsomes may be associated with the E-face particles in the thylakoid. These ultrastructural findings, in conjunction with those showing that PBsomes transfer excitation energy preferentially to PS II, have supported the hypothesis that the Eface particles represent PS II units in the thylakoid membrane. Thus far, the evidence supporting this hypothesis is based on ultrastructural observations of organisms deficient in some photosynthetic function (e.g. heterocysts, which lack PS II (Giddings & Staehelin, 1979), or mutants without PBsomes (Wollman, 1979)) and comparison with the ultrastructure of vegetative cells or wild-type organisms. Furthermore, these studies have not addressed the possible identity or function(s) of the P-face particles.

This study was undertaken in order to correlate the intramembranous thylakoid particles with photosynthetic functions by isolating fractions enriched in PS I and PS II from thylakoid membranes and characterizing the particles seen after freeze-fracture. The combination of biochemical characterization of the fractions coupled with ultrastructural observations provides further evidence for the identity and function of intramembranous thylakoid particles.

The culture of Phormidium laminosum (0H-1-p, clone 1) used in this study was a generous gift from Dr R. W. Castenholz. Cells were grown in medium D of Castenholz (1969) in a 14-1 New Brunswick Scientific Co. fermentor at 45°C with cool white fluorescent light at an intensity of 6 W m−2. The cells were aerated with 1% CO2 in air.

Cells were harvested at late log phase with a CEPA continuous flow centrifuge and used immediately. PS I and PS II fractions were isolated as described by Stewart & Bendall (1979). Phycobilisomes were isolated from P. laminosum, using the procedure described for Nostoc sp. (Troxler, Greenwald & Zilinskas, 1980).

Rates of electron transport from water to ferricyanide/2,6-dimethyl-p-benzoquinone (FeCy/DMBQ) and from dichlorophenolindophenol (DCPIP)/ascorbate to methyl viologen (MV) were measured at 25°C at light saturation (106 ergscm− 2 s−1) with a Clark-type oxygen electrode. Chlorophyll concentration was determined from 80% (v/v) acetone extracts according to Arnon (1949).

Absorption spectra were measured with a Cary 17D spectrophotometer. Corrected fluorescence emission spectra at 77 K were measured with an SLM 4800s spectrofluorometer. The excitation and emission slits were 8 nm and 2 nm, respectively. Samples were suspended in ‘buffer C’ (10 mM-HEPES/NaOH, pH7·5, 5 mM-NaH2PO4/K2HPO4, pH7·5, 10mM-MgCl2, 25% (v/v) glycerol (Stewart & Bendall, 1979)) at a chlorophyll concentration of 3μgml−1. For absorption measurements of chlorophyll proteins excised from a polyacrylamide gel, small pieces of gel containing the chlorophyll protein were placed in a 1 cm pathlength cuvette filled with distilled water and pressed flat against the front surface. A piece of gel containing no protein was placed in a matched cuvette to serve as a reference. The same gel slices were frozen in liquid N2, placed in a Dewar flask, and used for fluorescence measurements.

Chlorophyll protein complexes were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) according to Markwell, Reinman & Thornber (1978). Samples of thylakoid membranes were adjusted to a chlorophyll concentration of l·0mgml−1 and treated with 0·35% (w/v) lauryldimethylamine oxide (LDAO) for 30 min at 4°C. The treated membranes and PS I and PS II enriched fractions were suspended to a chlorophyll concentration of 0·4mgml−1 in a buffer containing 2·5 mM-Tris, 20mM-glycine (pH 8·4), and treated at 4°C with SDS at a final concentration of 0·8% (w/v) (SDS:chl = 20); 25 μl (10μg chlorophyll) was applied to the gel. Electrophoresis was carried out at 4°C for 3h at a constant voltage of 100 V. The unstained gels were scanned at 672 nm with a Gilford spectrophotometer equipped with a linear transport device. Total polypeptide composition of thylakoids and PS I and PS II fractions was determined as described previously (Glick & Zilinskas, 1982). The resolving gel was a 10% to 20% linear gradient of acrylamide. Samples were treated with SDS at 25°C. A 15 μg sample of chlorophyll was applied to each lane of the gel.

Intact filaments of P. laminosum were prepared for freeze-fracture electron microscopy by adding 5 ml of 70% (v/v) glycerol, in the growth medium (medium D), to an equal volume of cells that were washed and resuspended in medium D. The glycerol was added dropwise over a period of 1 h with stirring at 4°C. After at least 1 h in 35% glycerol, filaments were centrifuged at 3000 g for 5 min. Drops of PS I and PS II fractions in buffer C containing 35% glycerol and treated filaments (all at 4°C) were placed on copper support discs, frozen in liquid Freon cooled by liquid N2, and stored under liquid N2. All of these samples were fractured at − 100°C and etched for 2-3 min in a Balzers BAF-301 freeze-etch apparatus. In addition, buffer C containing 35% glycerol was also fractured and etched as a control and, as expected, no particles were detected. Replicas were examined in a Siemens 1A electron microscope operated at 80 kV. Magnifications were calibrated with a line grating replica. Micrographs for particle size measurements were made with a 7× magnifier equipped with a scale calibrated in 0·l-mm units. Particle diameters were measured according to Staehelin (1979). The width of a shadow of a given particle was measured over the shadowed half of the particle. Where the edge of the particle appeared fuzzy or irregular, a minimum width was always taken. In cases where shadows of adjacent particles overlapped, measurements were not made. Following particle size measurements, the mean particle diameter (ݲ), standard deviation (s), and standard error of the mean (Sݲ) were calculated for E-face and P-face particles and for particles in freeze-fractured PS I and PS II fractions. To determine whether differences in the mean particle diameters were statistically significant, the 99% confidence intervals for each mean were calculated.

Determining the structural sites of the two pigment systems and two photochemical reactions in red algal and cyanobacterial thylakoids has been the aim of several ultrastructural studies (Wollman, 1979; Giddings & Staehelin, 1979; Giddings et al. 1983). While valuable information has been obtained from these studies, a more definitive approach would be to correlate specific biochemical and physiological functions with ultrastructural observations. We have isolated PS I and PS II fractions from thylakoid membranes of Phormidium laminosum in order to show a more direct relationship between photosynthetic electron transport activities and intramembranous thylakoid particles in cyanobacteria.

One of the problems that has prevented isolation of photochemically active fractions from algal thylakoid membranes is the sensitivity of some photosynthetic functions to membrane solubilization techniques. PS II enriched fractions in particular have proven to be difficult to isolate without oxygen evolution. Recent developments in membrane fractionation have led to improved methods for the separation of photosynthetically active complexes from cyanobacterial thylakoid membranes (Stewart & Bendall, 1979). Fractionation techniques applicable to thermophilic organisms have also been developed in the belief that the physiological processes of these organisms would be less labile and therefore increase the probability of isolating membrane fractions that retain physiological functions (Newman & Sherman, 1978; Stewart & Bendall, 1979). It was for these reasons that the thermophilic cyanobacteriumP. laminosum was chosen for the studies reported here. The procedure of Stewart & Bendall (1979, 1980) used in this study was particularly useful as it yields a satisfactory separation of PS I and PS II, and each fraction can be recovered from the same experiment.

The photosynthetic electron transport activities of thylakoid membranes isolated from P. laminosum and those of the PS I and PS II enriched fractions are shown in Table 1. Although there is some residual PS I activity in the PS II fraction, the rate of oxygen evolution was as much as fivefold higher in the PS II fraction relative to intact membranes. The PS I fraction was also greatly enriched in PS I activity. The ratio of PS II to PS I activity in the intact membranes was 0·65, while in the PS I fraction the ratio was 0·13 (a fivefold difference relative to the membranes); in the PS II fraction the ratio was 5·27 (an eightfold difference relative to the membranes). In the best single experiment, the PS I and PS II activities constituted 89% and 85% of the total electron transport activity of their respective fractions.

Table 1.

Photochemical activities of isolated thylakoids and the PS I and the PS II enriched fractions

Photochemical activities of isolated thylakoids and the PS I and the PS II enriched fractions
Photochemical activities of isolated thylakoids and the PS I and the PS II enriched fractions

The spectral properties of the membranes and isolated fractions are shown in Fig. 1. The room temperature absorption maxima of membranes and the PS I fraction are at 678– 679 nm, while the PS II fraction has a maximum at 672 nm. These absorption maxima are consistent with published reports, which show that chlorophylls that absorb longer wavelengths are characteristic of the PS I-associated antenna, while PS II chlorophylls absorb maximally at wavelengths that are blue-shifted with respect to PS I chlorophylls (French, Brown & Lawrence, 1972). Furthermore, these absorption properties are similar to those reported for purified PS I and PS II fractions from spinach chloroplasts (Satoh & Butler, 1978).

Fig. 1.

Spectral properties of isolated thylakoid membranes. PS I fraction, and PS II fraction. A. Room temperature absorption spectra of isolated thylakoids (—), PS I fraction (‐ ‐ ‐), PS II fraction (‐· ‐). Chlorophyll concentrations of thylakoids, PS I fraction, and PS II fraction were l3·6, 11·7 and 9·5 μg chlorophyll ml−1, respectively, all suspended in buffer C. B. LOW temperature (77 K) fluorescence spectra of isolated thylakoids (—), PS I fraction (‐ ‐ ‐), PS II fraction (‐· ‐). All samples contained 3·0 μg chlorophyll ml−1, suspended in buffer C. Excitation was at 430.

Fig. 1.

Spectral properties of isolated thylakoid membranes. PS I fraction, and PS II fraction. A. Room temperature absorption spectra of isolated thylakoids (—), PS I fraction (‐ ‐ ‐), PS II fraction (‐· ‐). Chlorophyll concentrations of thylakoids, PS I fraction, and PS II fraction were l3·6, 11·7 and 9·5 μg chlorophyll ml−1, respectively, all suspended in buffer C. B. LOW temperature (77 K) fluorescence spectra of isolated thylakoids (—), PS I fraction (‐ ‐ ‐), PS II fraction (‐· ‐). All samples contained 3·0 μg chlorophyll ml−1, suspended in buffer C. Excitation was at 430.

The fluorescence emission at 77 K of isolated thylakoids has minor peaks at 684 and 694nm and a major peak at 727 nm. The fluorescence emission of the PS I fraction is at 726 nm, and the PS II fraction has major peaks at 684 and 694 nm and a lower peak at 724 nm. As indicated by the fluorescence spectrum of isolated membranes, the 727 nm peak is the major emission peak, because at 77 K the fluorescence yield of and the energy transferred to the PS I chlorophyll is greater than that of PS II chlorophyll (Goedheer, 1972). Therefore, the emission at 724 nm in the PS II fraction represents the contribution of only a small amount of PS I chlorophyll present in the PS II fraction. In cells of red algae and cyanobacteria, the fluorescence emission peaks at 77 K due to chlorophyll a are usually observed at or near 685, 695 and between 710 and 73Onm (Rijgersberg & Amesz, 1980). The emission at 685 and 695nm has been attributed to PS II and the 710—73Onm emission to PS I (Diner & Wollman, 1979; Rijgersberg & Amesz, 1980).

When isolated thylakoids and the PS I and PS II fractions are solubilized with SDS (SDS: chi (w/w) = 20) and electrophoresed in a 5% polyacrylamide gel at 4°C, the chlorophyll remains non-covalently bound to its apoprotein, so that it is possible to resolve the chlorophyll proteins and visualize them in the gel (Markwell et al. 1978). The chlorophyll proteins may then be excised from the gel and their spectral properties investigated. Two chlorophyll proteins are resolved from isolated thylakoid membranes. The spectral properties of each of them (Fig. 2) indicate that the chlorophyll protein of lower electrophoretic mobility is associated with PS I, as its room temperature absorption and 77 K fluorescence emission maxima, 676 and 723 nm, respectively, are characteristic of PS I chlorophyll, while the faster migrating chlorophyll protein (absorption and fluorescence maxima at 670 and 686 nm, respectively) is characteristic of PS II chlorophyll.

Fig. 2.

Spectral properties of chlorophyll proteins separated on a 5% polyacrylamide gel. A. Room temperature absorption spectra of PS I (—) and PS II (‐ ‐ ‐) chlorophyll proteins. Full scale was 0·5 for the PS II chlorophyll protein spectrum and l·0 for the PS I chlorophyll protein spectrum. B. LOW temperature (77 K) fluorescence spectra of PS I (—) and PS II (‐ ‐ ‐) chlorophyll proteins. Excitation was at 430 nm.

Fig. 2.

Spectral properties of chlorophyll proteins separated on a 5% polyacrylamide gel. A. Room temperature absorption spectra of PS I (—) and PS II (‐ ‐ ‐) chlorophyll proteins. Full scale was 0·5 for the PS II chlorophyll protein spectrum and l·0 for the PS I chlorophyll protein spectrum. B. LOW temperature (77 K) fluorescence spectra of PS I (—) and PS II (‐ ‐ ‐) chlorophyll proteins. Excitation was at 430 nm.

The pattern of chlorophyll proteins that is resolved when the PS I and PS II fractions are run in the 5% gel at 4°C reveals the presence of predominantly the PS I-associated chlorophyll protein in the PS I fraction (Fig. 3), while the PS II sample shows a fourfold enrichment of PS II chlorophyll protein relative to the amount present in the membranes. In the membranes, 85% of the total chlorophyll applied to the gel is associated with the PS I chlorophyll protein and 15% with the PS II chlorophyll protein (Fig. 3). In the PS I fraction, 99% of the protein-associated chlorophyll is PS I chlorophyll, while 58% of the chlorophyll of the PS II sample is associated with PS II chlorophyll protein. These data may overestimate the amount of PS I chlorophyll protein relative to PS II, as the PS II chlorophyll protein complex is much more susceptible to dissociation of the chlorophyll from the apoprotein than is the PS I complex. The small peak in the gel scan at 4—5 cm represents detergent-solubilized chlorophyll that is not associated with protein and runs with the dye front during electrophoresis. These electrophoretic patterns are comparable to those reported by Stewart (1980) using a similar gel system, except that in the work reported here, very little PS II chlorophyll protein was detected in the PS I sample.

Fig. 3.

Profiles of chlorophyll proteins separated on a 5% polyacrylamide gel at 4°C according to Markwell et al. (1978). A 10μg sample of each of: A, isolated thylakoids; B, PS I; and c, PS II fractions were applied to the gel (SDS: chi (w/w) = 20). Thylakoids were pre-treated with LDAO at LDAO:chl (w/w) = 3·5 for 30min at 4°C. Unstained gels were scanned at 672 nm.

Fig. 3.

Profiles of chlorophyll proteins separated on a 5% polyacrylamide gel at 4°C according to Markwell et al. (1978). A 10μg sample of each of: A, isolated thylakoids; B, PS I; and c, PS II fractions were applied to the gel (SDS: chi (w/w) = 20). Thylakoids were pre-treated with LDAO at LDAO:chl (w/w) = 3·5 for 30min at 4°C. Unstained gels were scanned at 672 nm.

Re-electrophoresis of the chlorophyll proteins in a Laemmli system (SDS: chi = 100) at room temperature has revealed that the PS I chlorophyll protein consists of a major polypeptide with a molecular weight of 65×103 and minor polypeptides of molecular weight 10-18× 103 (data not shown). These polypeptides are of similar size to those of a purified PS I reaction centre from a thermophilic cyanobacterium (Nechushtai et al. 1983). The PS II chlorophyll protein was made up of two polypeptides of molecular weight 51 × 103 and 47 × 103, which are similar in size to polypeptides of PS II chlorophyll protein from other cyanobacteria (Rusckowski & Zilinskas, 1980; Guikema & Sherman, 1983). In Fig. 4, it can be seen that the PS I fraction contains a 65×103Mr polypeptide and that the PS II fraction shows an enrichment of two polypeptides of 52 and 48×103Mr. These results indicate that the PS I fraction is free of polypeptides representing PS II chlorophyll protein and that the PS II fraction is highly enriched in PS II chlorophyll protein polypeptides.

Fig. 4.

Polypeptides of isolated thylakoids, PS I and PS II fractions separated on a linear gradient (10% to 20%) of acrylamide. Fifty microlitres of sample buffer (Glick & Zilinskas, 1982) was added to 15 μl of each sample (all at a chlorophyll concentration of 1·0 mgml−1). The entire sample (15 μg chlorophyll) was applied to the gel. Samples were treated with SDS at room temperature. Lane 1, P. laminosum PBsomes; lane 2, isolated thylakoids; lane 3, PS I fraction; lane 4, PS II fraction; lane 5, molecular weight standards: phosphorylase b (94×103), bovine serum albumin (68×103), ovalbumin (43×103), carbonic anhydrase (30×103), soybean trypsin inhibitor (21×103), lysozyme (14×103).

Fig. 4.

Polypeptides of isolated thylakoids, PS I and PS II fractions separated on a linear gradient (10% to 20%) of acrylamide. Fifty microlitres of sample buffer (Glick & Zilinskas, 1982) was added to 15 μl of each sample (all at a chlorophyll concentration of 1·0 mgml−1). The entire sample (15 μg chlorophyll) was applied to the gel. Samples were treated with SDS at room temperature. Lane 1, P. laminosum PBsomes; lane 2, isolated thylakoids; lane 3, PS I fraction; lane 4, PS II fraction; lane 5, molecular weight standards: phosphorylase b (94×103), bovine serum albumin (68×103), ovalbumin (43×103), carbonic anhydrase (30×103), soybean trypsin inhibitor (21×103), lysozyme (14×103).

Electron micrographs of freeze-fractured cells of P. laminosum (Fig. 5) reveal the two types of fracture faces present in the thylakoid membranes. Unlike the situation found in higher plant chloroplasts, in P. laminosum the intra-thylakoid space is extremely narrow, making the steps between surfaces and fractures nearly imperceptible. The arrowheads in Fig. 5 illustrate areas of transition between the surface and the fracture face of the membrane. The E-face is characterized by fewer, widely spaced particles, while the P-face is densely packed with particles. The mean diameters of the particles on the E-face and P-face are 8·3nm and 7·lnm, respectively. The main size class of E-face particles is 8·Onm, while most of the P-face particles are either 6·0 or 7·0nm in diameter. The E-face particles are sometimes seen to be organized into short rows a few particles long (Fig. 5), similar to these described by Giddings et al. (1983). Long rows of particles were seen only infrequently.

Fig. 5.

Freeze-fracture micrograph of a portion of a P. laminosum cell showing crossfractured thylakoids (t), and exposed particles on the E- and P-faces of the membranes. Bar, 0·25 μm.

Fig. 5.

Freeze-fracture micrograph of a portion of a P. laminosum cell showing crossfractured thylakoids (t), and exposed particles on the E- and P-faces of the membranes. Bar, 0·25 μm.

Electron micrographs of freeze-fractured PS I and PS II fractions are shown in Figs 6 and 7. The particles in the PS I fraction are in packed arrays that appear to be associated with membrane. The general distribution of the particles is qualitatively similar to the distribution of the particles on the P-face of the P. laminosum cells, i.e. many particles in close proximity to each other. The particles in the PS II fraction are not associated with membrane but are scattered freely in the suspending medium. Occasionally, groups of particles can be seen that appear to form a short row. The mean sizes of the particles in the PS I and PS II fractions are 7·8 and 8·7 nm, respectively, and the range of particle sizes is shown in Fig. 8 in the form of a histogram. The PS II particle histogram is superimposed over that of the E-face particle histogram, as are the PS I and P-face particle histograms. The maxima of both the E-face and PS II histograms are at 8·Onm. The P-face particle histogram has a broad maximum at 6·0—7·0nm, while that of the PS I histogram is at 7·0 nm. There is good agreement between the E-face and PS II histograms. Statistical analysis shows (Table 2 and Fig. 9) that the difference between the mean diameter of E-face and PS II particles is not significant, indicating that the particles seen in the freeze-fractured PS II preparation are the same as those on the E-face of freeze-fractured P. laminosum cells. There were, however, significant differences between the mean E-face and P-face particle sizes and the mean PS I and PS II particle sizes. The P-face and PS I particle histograms have the same basic shape; the significant difference in their mean particle size is probably a result of the fact that the entire PS I particle population is ‘shifted’ l·0nm higher than the P-face particle population of thylakoid membranes. This suggests that in addition to PS I, there are other components that contribute to the particles in the thylakoid P-face.

Table 2.

Statistical analysis of freeze-fracture particles of P. laminosum thylakoids and PS I and PS Il-enriched fractions

Statistical analysis of freeze-fracture particles of P. laminosum thylakoids and PS I and PS Il-enriched fractions
Statistical analysis of freeze-fracture particles of P. laminosum thylakoids and PS I and PS Il-enriched fractions
Fig. 6.

Freeze-fractured PS I fraction. Bar, 0·25 μm.

Fig. 6.

Freeze-fractured PS I fraction. Bar, 0·25 μm.

Fig. 7.

Freeze-fractured PS II fraction. Bar, 0·25 μm.

Fig. 7.

Freeze-fractured PS II fraction. Bar, 0·25 μm.

Fig. 8.

Histograms of particle sizes on E- and P-faces, and in PS I and PS II fractions. Number of particles measured for all classes was 560.

Fig. 8.

Histograms of particle sizes on E- and P-faces, and in PS I and PS II fractions. Number of particles measured for all classes was 560.

Fig. 9.

Comparison of the mean particle diameters of particles on the E- and P-faces of P. laminosum thylakoids and particles in freeze-fractured PS I and PS II preparations. The 99% confidence intervals for each mean particle size are indicated.

Fig. 9.

Comparison of the mean particle diameters of particles on the E- and P-faces of P. laminosum thylakoids and particles in freeze-fractured PS I and PS II preparations. The 99% confidence intervals for each mean particle size are indicated.

It is nearly certain that the P-face of cyanobacterial thylakoids contains more than just PS I particles. Arntzen (1978) proposed that on the P-face of spinach thylakoids there are particles whose functions are not related to PS I. It was argued that this must be the case since the ratio of P-face to E-face particles in higher plant mature grana thylakoids is 3:1 (Staehelin, 1979), while the ratio of PS I to PS II reaction centres is about 1:2 (Melis & Brown, 1980). In spinach thylakoids, the P-face has two size classes of particles that average 8·0—8·5nm and 10·5nm in diameter (Staehelin, 1979; Armond et al. 1977,). Furthermore, purified coupling factor complexes and isolated cytochrome b/f complèxes reconstituted into liposomes have shown particles of diameters 9·5nm (Mullet, Pick & Arntzen, 1981) and 8·5nm (Morschel & Staehelin, 1983), respectively. Kaplan & Arntzen (1982) have therefore suggested that the particles on the P-face of spinach thylakoids represent a mixture of PS I complexes, cytochrome b/f complexes and the intrinsic membrane component of chloroplast coupling factor (CFo).

Although less is known about the thylakoid ultrastructure in cyanobacteria, similarities between the complexes in cyanobacterial thylakoids and those in the chloroplast membranes have been documented (Staehelin et al. 1978). In addition, a cytochrome b/f complex has been isolated from cyanobacteria (Krinner, Hauska, Hurt & Lockau, 1982), using a procedure similar to that used with spinach chloroplasts, implying that the complex probably exists as a discrete membrane particle, as has been shown for spinach thylakoids (Morschel & Staehelin, 1983). Unfortunately, particle sizes for the cyanobacterial cytochrome b/f complex (or CFQ) are not known.

A further indication that there are particles other than PS I complexes on the P-face of cyanobacterial thylakoids is seen when comparing the ratio of P-face to E-face thylakoid particles and the ratio of PS I to PS II reaction centres. The ratio of P-to E-face particles in various cyanobacteria has been reported to be greater than 5 (Armond & Staehelin, 1979; Giddings & Staehelin, 1979; Coxeta/. 1981), while the ratio of PS I to PS II reaction centres is 2·3-2·5 (Kawamura, Mimuro & Fujita, 1979; Myers, Graham & Wang, 1980; Melis & Brown, 1980; Manodori & Melis, 1984), indicating that not all P-face particles represent PS I complexes.

In addition to PS I, cytochrome b/f and CF0 complexes, there may be particles in thylakoid membranes that are related to respiratory functions. The site of respiratory electron transport in cyanobacteria has not been established, but most evidence suggests that the respiratory chain is located in both the thylakoid and the plasma membrane. If this is so, then some of the thylakoid P-face particles might function in respiratory electron flow (Binder, 1982). The respiratory and photosynthetic electron transport chains do contain some of the same components such as quinones, the cytochrome b/f complex, plastocyanin and cytochrome C553 (Binder, 1982). Peschek (1983) has shown that the cytochrome b/f complex participates in both respiratory and photosynthetic electron transport, implying a close physical relationship of respiration and photosynthesis in cyanobacteria. He proposed (and Sandmann & Malkin (1983) have confirmed) that the cytochrome b/f complex can donate electrons to either P700 or cytochrome oxidase in cyanobacteria. In the light of these findings, it may be proposed that particles representing respiratory complexes in the thylakoid membrane are in close association with PS I complexes, and that the P-face of the freeze-fractured thylakoid membranes may therefore contain particles with dual roles in photosynthesis and respiration, as well as particles involved in either function alone.

Consequently, as the P-face of thylakoid membranes probably contains more than just PS I complexes, one must assume that at least some of these complexes must have been removed as a consequence of the PS I isolation procedure. This is the most logical explanation to account for differences in the mean particle sizes of the thylakoid P-face and the isolated PS I fraction. These smaller, non-PS I, P-face particles might have been partially solubilized by the detergent and separated from the PS II complexes during subsequent chromatography on Sepharose 6B. Unfortunately, owing to dense packing, similar sizes, and diverse functions of P-face particles, their identification has been slow; it is hoped that, with further characterization of other detergent-solubilized functional complexes of cyanobacteria by both biochemical and microscopic means, there will be less need for speculation.

As noted above, the PS I freeze-fracture particles appear to be membrane-bound but the PS II particles are not. Such being the case, an explanation may be offered as to how the separation of PS II from PS I occurs. In the membrane fractionation procedure, the solubilization of the thylakoid membranes with LDAO is followed by ultracentrifugation (100000 g for lh). The pellet fraction of the centrifugation contains the bulk of the chlorophyll and the greater PS I activity (PS I fraction), while the clarified green supernatant shows the higher rate of oxygen evolution (PS II fraction). This supernatant can be pelleted after it has been passed through a Sepharose 6B column, which removes the detergent and residual phycobiliprotein. The fact that ultracentrifugation separates PS II from PS I implies that the PS II fraction consists of rather small particles, since they remain in the supernatant after being subjected to 100 000 g for 1 h and require centrifugation at 100 000g for 16h to sediment them. This also implies that LDAO is selectively solubilizing PS II from the membrane but leaves PS I in the membrane.

A small amount (10–20%) of membranous material was observed in freezefracture replicas of the PS II fraction. There were also particles present on some of the membranous material with diameters of about 7·5 nm (data not shown). In view of the above discussion about the nature of the PS I particles being membranebound, it is probable that the membranous material in the PS II fraction is the source of the PS I contamination of this fraction. As these clearly different particles were not included in measurements made, a true representation of the PS II particles was obtained.

The observation of rows of particles on the E-face of thylakoids in PBsome-containing organisms has led many investigators to propose that the E-face particles are structural equivalents of PS II centres in the membrane (Neushul, 1971; Lefort-Tran etal. 1973; Lichtle & Thomas, 1976; Wollman, 1979; Golecki, 1979; Giddings & Staehelin, 1979). A more rigorous study of the relationship between thylakoid E-face particles and PBsomes has been published (Giddings et al. 1983) in which the spacing both within and between E-face particle rows and PBsomes was compared. The authors concluded that there was a close correspondence between rows of E-face particles and PBsomes but noted that rows of E-face particles were not consistently seen in the thylakoids. The work presented here supports the observation that rows of E-face particles are not a predominant feature of the E-face of cyanobacterial thylakoids. It seems likely that rows of E-face particles do occur in some cyanobacterial species, as they have been observed by many investigators, but they are not universally found in cyanobacteria or red algae (Staehelin et al. 1978). The question of their significance still exists.

This work was supported in part by the Science and Education Administration of the United States Department of Agriculture under grant 85-CRCR-1-1562 from the Competitive Research Grants Office. New Jersey Agricultural Experiment Station, Publication no. D-01104-1-85, supported by State Funds and by the United States Hatch Act. Thanks are given to Charles Kupatt for assistance with the statistical analysis.

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