J. Cell Sci. 10, 3O7-3H (1972) 307

Printed in Great Britain

FINE STRUCTURE OF SWARMERS OF

CLADOPHORA AND CHAETOMORPHA

II. THE CHLOROPLAST

D. G. ROBINSONAstbury Department of Biophysics, The University of Leeds, Leeds, LSz gJT, England

SUMMARY

Freeze-etched chloroplast thylakoids in swarmers of Chaetomorpha reveal the in situ arrange-ment of these thylakoids and also a completely new fracture face. This face, which is com-pletely smooth, does not bear any resemblance to any freeze-etched fracture face previouslyrecorded. The relative merits of thylakoid models are discussed in relation to this new fractureface.

INTRODUCTION

The substructure of the chloroplast thylakoid as revealed by freeze-etching hasprimarily been investigated using higher plants such as spinach. Whilst the viewsshown by both Miihlethaler, Moor & Szarkowski (1965) on the one hand and Park &Branton (1966), Branton & Park (1967), Park & Pfeifhofer (1969a) on the other handare more or less identical, the interpretations differ. The view that the fracture planefollows the interface between membrane and medium, sustained by Miihlethaler'sgroup, has received little corroborative evidence since 1965; whereas Park's grouphave not only shown that membranes may split during the freeze-fracturing process(Branton, 1966) but by application of deep-etching Park & Pfeifhofer (1969a) havedemonstrated a relatively smooth face external to the faces revealed by conventionalfreeze-etching.

Examples of freeze-etched chloroplast thylakoids in situ as opposed to those obtainedby homogenizing spinach leaves are few, and certainly no new features regarding thestructure of the thylakoid have been observed. Although the arrangement of thylakoidsmay differ between algal and higher plant cells, the structure of the thylakoid may beexpected to be similar. In this second paper on the fine structure of swarmers of theCladophorales the freeze-etched views of the chloroplast of Chaetomorpha swarmersare shown. As described previously for spinach thylakoids, 2 types of particulate faceexist in these swarmers, but a new fracture face is also apparent. This face, which iscompletely smooth and is not related to the face revealed by deep-etching, is arepeating one. An attempt at allocation of this face to thylakoid models alreadyproposed is made.

MATERIALS AND METHODS

These were precisely as described in the first paper of this series (Robinson & Preston, 1971).

308 D. G. Robinson

RESULTS

The chloroplast substructure, and the relationship of the chloroplast thylakoids tothe rest of the cell components, is shown in Fig. i. The stroma (sr) and grana (gr)regions are evident. Numerous thylakoids are present, some cross-fractured and someseen in surface views. The thylakoids are presented at higher magnifications in Figs.2-5. Unlike the views presented by Branton & Park (1967), Park & Branton (1966),Muhlethaler (1967) and Miihlethaler et al. (1965), these thylakoids reveal 4 faces,one of which has not been documented before:

(1) A predominant face carrying loosely distributed particles approximately 12 nmin diameter - face B.

(2) A face with smaller particles, diameter 6 nm, more densely packed and often inregular arrays - face A.

(3) A face bearing both large and small particles on it - face C. It was found verydifficult to assign this face; indeed in this case the existence of a real C face isdoubtful.

(4) A face completely smooth bar a very few scattered particles 12 nm diameter -face D.

The letters A, B, C are used following Park & Branton (1966). Recently Park &Pfeifhofer (1969 a) were able to demonstrate the presence of a relatively smoothsurface by deep-etching of fractured, isolated, spinach thylakoids. A small number ofparticles 10-12 nm diameter exist on this ' new face' and were later (Park & Pfeifhofer,19696) shown to correspond to Ca2+-dependent ATPase. Face D shown in Figs. 3-5does not appear to be even superficially like the view presented by Park & Pfeifhofer;it is almost completely smooth with only very rare particles. This observation is nota freak aberration since, as can be seen in Fig. 4, face D is a regularly repeatingstructure amongst appressed thylakoids.

The spatial sequence of faces is very difficult to ascertain. Fig. 2 shows face A tooccur immediately above face C, whilst Fig. 4 is marked to show repeating A, B andD faces. Fig. 3 shows (arrow 1) what appears to be a sequence of A-D-B faces; Fig. 5shows a sequence of C-A—B-D faces. Fig. 4 also shows an A—B-D sequence andseveral A-D sequences with a deep ridge between the A and D faces. Measurementof thylakoid thickness as 12-6 nm is in agreement with other freeze-etch results and itseems that the inner membrane surface (determined from those thylakoids whichmerge into the stroma, e.g. Fig. 5, arrow /) whilst being separated from otherthylakoids, is particulate, but the designation of this as an A, B or C face is somewhatdifficult. If these deductions are correct then they would conform better with Park &Branton's (1966) concept than with that of Muhlethaler (1967). This means thatfaces A and B (and C) occur within the thylakoid membrane.

DISCUSSION

The new observation in chloroplast thylakoids of a naked face (face D) deservessome comment. As mentioned earlier, I feel I must rule out the possibility that it

Chloroplast of swarmers 309

corresponds to the face revealed by deep-etching as in Park & Pfeifhofer (1969 a).Other plant cell membranes have been shown by Branton (1966) to split open intothe 2 constituent bi molecular leaflets by the freeze-fracturing process, revealinggranules on the inner membrane surfaces. If face D in the thylakoids here represents acleavage through the hydrophobic areas of the thylakoid membrane, this would be inapposition to the Branton & Park (1967) model, since such a split cannot occur withoutrevealing a particulate face. On the other hand, thylakoid substructure according toMiihlethaler et al. (1965) is to be based on the unit-membrane concept (with particleson the outer hydrophilic. surfaces) and they have maintained that the cleavage planedoes not go through the centre of the unit membrane.

One difference between the models of these 2 groups of workers is the size of theparticles associated with the fracture faces. Using spinach chloroplasts Branton &Park (1967) measure the large particles as 17-5 x 9 nm and the small ones at 11 nm,whilst Miihlethaler et al. (1965) with the same material show large particles of averagesize 12x6 nm and small ones of 6 nm. This is a fundamental difference which shouldbe removed. Three solutions are possible; first, that one group of authors hasmeasured the particles incorrectly; secondly, different growth conditions for thespinach has produced different values; and, thirdly, there could be one basic particlesize with the particles existing in various degrees of fusion. The present results onChaetomorpha swarmers give particle sizes of 12 and 6 nm which correspond to thoseof Miihlethaler's group.

Conflict also arises from the nature of the membrane-bound particles. According toMiihlethaler & Wehrli (1969), the large particles correspond to Ca2+-dependentATPase particles which can be removed from the thylakoids by washing with EDTA.Earlier, Howell & Moudrianakis (1967a, b) had recognized that these particles do notparticipate in photoreductions. Miihlethaler & Wehrli also isolated carboxydismutaseparticles and after negative staining showed them to be 10-12 nm squares made up of4 subunits. They assign these particles to the outer face of the thylakoid membrane,and equate them to the ATPase particles. Furthermore, they criticize Branton & Park(1967) for assuming that the ridge so often present at the base of the membrane infreeze-etch preparations is the other half of the bimolecular leaflet. They argue thatwhen specimens are rapidly frozen in 20% glycerol, ice crystals are formed in theeutectic which have a thin glycerol lamella around them. Similar glycerol lamellaeare presumed to be formed at the interface of the specimen and the medium and formsmall ridges after fracturing. Deep etching makes these structures more pronouncedas glycerol will not sublime. Park and his co-workers (Park & Shumway, 1968; Park& Pfeifhofer, 1969 a, b) have overcome this criticism with regard to deep-etching byfreezing their specimens in water rather than in a glycerol solution. The presence ofan outer layer to the thylakoid after deep-etching must therefore represent a true outerlayer to the thylakoid. This new face is shown to have particles on it 10-12 nm indiameter. Here again Park's group differs from Miihlethaler's. Park shows these newparticles to be removed after EDTA washing whilst the large 17'5-nm diameterparticles remain. A similar demonstration was made for the smaller 11-nm particlesand their overlying layer. Miihlethaler & Wehrli (1969), on the other hand, claim that

310 D. G. Robinson

their large (12-nm) thylakoid particles are removed by EDTA washing. However, theydo not show any freeze-etch evidence of this, and it would appear that they haveassumed their large (freeze-etched) thylakoid particles to be identical with theATPase particles of Howell & Moudrianakis (1967 a, b).

Moudrianakis, Howell & Karu (1968) have shown the existence of 2 protein specieswhich were classified as ' quantasomes'. These 2 species were in fact carboxydismutaseand the Ca2+-dependent ATPase. They differed in extraction characteristics, molecularweight, electrophoresis movement, and in negative staining by which the former wasshown to be 12 nm and the latter 10 nm in diameter. Because of their size andphysiological action these authors claimed that the ATPase particles were the same asthe quantasomes of Park & Biggins (1964). Park & Pfeifhofer (1969a) suggest that thei7-5-nm particles seen by freeze-etching are only part of the whole quantasome andshould be called ' quantasome cores', which only seems to complicate matters further.

An attempt to rationalize the particulate nature of freeze-etched thylakoids withthe results obtained by negative staining has been made by Arntzen, Dilley & Crane(1969). Using function assays these authors were able to show the physical separationof photosystems I and II by the action of digitonin on blended spinach chloroplasts.That fraction enriched in photosystem II activity had predominantly i7'5-nm particleson its freeze-etched membrane faces whilst photosystem I was seen to have only11-nm particles. Although the intact chloroplast membranes were shown afternegative staining to have 10-11 nm particles associated with the outer membranesurface, neither of the digitonin fractions showed any negatively stained particles.Arntzen et al. (1969) have therefore put forward a schematic representation of thechloroplast thylakoid in which all possible faces of the thylakoid membrane areparticulate: the exterior faces bearing 2 types of particles which can be seen bynegative staining (carboxydismutase and ATPase), and interior faces bearing thefreeze-etch particles. Certainly this scheme has many advantages but no matter whichscheme is picked, a completely naked face as shown above does not fit. Moreover, therecent demonstration by Bretscher (1971), that the sialoglycopeptide of humanerythrocytes extends right through the cell membrane, with different parts of thepolypeptide chain residing on both inner and outer membrane surfaces, may beextended to membranes in general, providing support for the theory that membranessplit during freeze-etching revealing (probably) proteinaceous particles. If this is so,then a completely naked membrane face, albeit a thylakoid membrane, appears all themore strange.

Two possible answers are forthcoming: it is possible that the freeze-etched algalchloroplast differs in its thylakoid substructure from that of the higher plant; if so,then a naked face may be characteristic of such thylakoids. It seems more likely,however, that the naked areas shown are related to the developmental nature of thesystem. These free-swimming swarmers are a transient stage and soon settle, developa wall, enlarge and divide. It may well be that such naked faces are not permanently sobut are particulate faces on which particles have not yet developed.

Chloroplast of swarmers 311

I would like to thank the Science Research Council for a Postgraduate Award, D. Brain andL. Child for the photographic work and Professor R. D. Preston, F.R.S., for some usefulsuggestions.

REFERENCES

ARNTZEN, C. J., DILLEY, R. A. & CRANE, F. L. (1969). A comparison of chloroplast membranesurfaces visualized by freeze etch and negative staining techniques; and ultrastructuralcharacterization of membrane fractions obtained from digitonin treated spinach chloroplasts.J. CellBiol. 43, 16-31.

BRANTON, D. (1966). Fracture faces of frozen membranes. Proc. natn. Acad. Sci. U.S.A. 55,1048—1056.

BRANTON, D. & PARK, R. B. (1967). Subunits in chloroplast lamellae. .7. Ultrastruct. Res. 19,283-303.

BRETSCHER, M. S. (1971). Major human erythrocyte glycoprotein spans the cell membrane.Nature New Biology, Lond. 231, 229-232.

HOWELL, S. H. & MOUDRIANAKIS, E. N. (1967a). Hill reaction site in chloroplast membranes:non-participation of the quantasome particle in photoreduction.^. molec. Biol. 27, 323-333.

HOWELL, S. H. & MOUDRIANAKIS, E. N. (19676). Function of the 'quantasome' in photo-synthesis: structure and properties. Proc. natn. Acad. Sci. U.S.A. 58, 1261-1268.

MOUDRIANAKIS, E. N., HOWELL, S. H. & KARU, A. E. (1968). Characterization of the'quantasome' and its role in photosynthesis. In Comparative Biochemistry and Biophysics ofPhotosynthesis (ed. K. Shibata, A. Takamiya, A. T. Jagendorf & R. C. Fuller), pp. 67-81.Tokyo: University of Tokyo Press.

MOHLETHALER, K. (1967). The ultrastructure of the plastid lamellae. In Biochemistry ofChloroplasts, vol. 1 (ed. T. W. Goodwin), pp. 49-63. London and New York: AcademicPress.

MOHLETHALER, K., MOOR, H. & SZARKOWSKI, J. W. (1965). The ultrastructure of the chloro-plast lamellae. Planta 67, 305-323.

MOHLETHALER, K. & WEHRLI, E. (1969). Freeze-etch studies on photosynthetic lamellae. InProgress in Photosynthesis Research, vol. 1 (ed. H. Metzner), pp. 87-90. Tubingen: Inter-national Union of Biological Sciences.

PARK, R. B. & BIGGINS, J. (1964). Quantasome: size and composition. Science, N.Y. 144,1009-1011.

PARK, R. B. & BRANTON, D. (1966). Freeze-etching of chloroplasts from glutaraldehyde-fixedleaves. Brookhaven Symp. Biol. 19, 341-352.

PARK, R. B. & PFEIFHOFER, A. O. (1969a). Ultrastructural observations on deep-etchedthylakoids. J. Cell Sci. 5, 299-311.

PARK, R. B. & PFEIFHOFER, A. O. (19696). The effect of ethylenediaminetetra-acetate washingon the structure of spinach thylakoids. J. Cell Sci. 5, 313-319.

PARK, R. B. & SHUMWAY, L. K. (1968). The ultrastructure of fracture and deep etch faces ofspinach thylakoids. In Comparative Biochemistry and Biophysics of Photosynthesis (ed.K. Shibata, A. Takamiya, A. T. Jagendorf & R. C. Fuller), pp. 57-66. Tokyo: University ofTokyo Press.

ROBINSON, D. G. & PRESTON, R. D. (1971). Fine structure of swarmers of Cladophora and Chaeto-morpha. I. The plasmalemma and Golgi apparatus in naked swarmers. J. Cell Sci. 9, 581-601.

(Received 20 August 1971)

312 D.G.Robinson

All micrographs are printed with the metal shadowing direction running from top to bottom.Fig. i. Cross-fractured swarmer of Cliaetomorpha melagonium. Note the great extentto which the chloroplast fills the cell; gr, grana and sr, stroma regions, x n 800.Fig. 2. Surface fracture through a chloroplast thylakoid. One face (A) bears closelypacked particles 6 nm diameter, the other face (C) has a mixture of large 12-nmparticles with the smaller 6-nm particles, x 55200.Fig. 3. Surface fracture revealing 4 different faces. Faces A and C are as inFig. 2. Also present is a loosely particulate face (B) bearing 12-nm particles and acompletely naked face D. Arrow / indicates a sequence of A-D-B faces, x 73 200.

Chlorofrlast of swarmers

D. G. Robinson

Fig. 4. Note the repeating nature of the naked face, D. The lack of a ridge between Band D faces make it difficult to ascertain the regular repeat, x 73 200.Fig. 5. A sequence of C-A-B-D faces is present here. Note the occasional 12-nm par-

ticle on the otherwise naked (D) face, x 55 200.