J. Cell Sci. 46, 87-96 (1980) 87Printed in Great Britain © Company of Biologists Limited igSo

CHLOROPLAST DIVISION IN SPINACH

LEAVES EXAMINED BY SCANNING ELECTRON

MICROSCOPY AND FREEZE-ETCHING

N. CHALY, J. V. POSSINGHAM AND W. W. THOMSON*CSIRO Division of Horticultural Research, GPO Box 350,Adelaide, Australia

SUMMARY

Spinach leaf disks were cultured for 5 days in low-intensity green light and then were trans-ferred to high-intensity white light. Harvests over the next 16 h established that cell areaincreased by about 80% and chloroplast number per cell increased by about 65 %, while thepercentage of dumbbell-shaped chloroplasts per cell decreased by 65 %. Freeze-etch replicasof fixed and unfixed leaf disks, as well as scanning electron-microscope preparations of fixedmaterial, contained dumbbell-shaped chloroplasts constricted to various degrees. Freeze-etchreplicas of unfixed cells from young leaf bases, in which the number of chloroplasts per cell isknown to be rapidly increasing, also contained many constricted chloroplasts. It is concludedthat dumbbell-shaped chloroplasts occur in vivo and represent a stage in the division ofchloroplasts.

INTRODUCTION

During the normal growth of higher plant leaves a massive increase occurs in thechloroplast population. Chloroplasts in large numbers are required for the largeincrease in the chloroplast population per cell which occurs during cell expansion andto supply chloroplasts to new cells arising by cell division (Possingham, 1980). It hasbeen suggested for a long time, and is now generally accepted, that these organellesincrease in number by the division of pre-existing plastids (Schimper, 1885; Kirk &Tilney-Bassett, 1978). In lower plants, a sequence in which 1 chloroplast passesthrough a dumbbell stage to give 2 daughter chloroplasts has been convincinglydocumented by cinematography (Green, 1964). In higher plants, however, althoughchloroplast profiles with central constrictions have been observed in a number ofspecies by light and electron microscopy, evidence that dumbbell-shaped chloroplastsactually divide is largely circumstantial (Lance, 1958; Senser & Schotz, 1964; Cran &Possingham, 1972; Esau, 1972; Leech, Rumsby & Thomson, 1973; Whatley, 1974).Nevertheless, in both spinach and tobacco (Boasson & Laetsch, 1969; Possingham &Saurer, 1969) and more recently in wheat (Boffey, Ellis, Sellden & Leech, 1979) andin bean (Whatley, 1980), microscope observations of developing leaf cells haveestablished that there is a correlation between the increase in chloroplast number andthe presence of dumbbell-shaped chloroplasts.

In the present work we have applied scanning electron microscopy (SEM) and

• Department of Botany, University of California, Riverside, California, USA.

88 N. Chaly, J. V. Possingham and W. W. Thomson

freeze-etching to spinach leaf tissues to obtain more detailed information on the ultra-structure and 3-dimensional appearance of their chloroplasts during division. Weexamined cells from the base of young spinach leaves at a stage when chloroplastnumbers are known to be rapidly increasing (Possingham & Saurer, 1969). We alsoused the cultured leaf disk system (Possingham & Smith, 1972), in which the pro-portion of dumbbell-shaped chloroplasts is increased by culturing tissue con-secutively at different light intensities.

MATERIALS AND METHODSSpinach (Spinacea oleracea L.) plants were grown in aerated nutrient solution in a growth

cabinet as in earlier experiments (Possingham & Saurer, 1969). Disks were excised from thebases of 2-cm-long spinach leaves and were cultured on nutrient agar plates as previouslydescribed (Possingham & Smith, 1972). The plates were kept in continuous green light(60 fiE m~* s"1) for 5 d and were then transferred to continuous white light (300 fiE m~' s"1)for 0-24 h (to-tu).

Light microscopy. Sections approximately 100 /tm thick were cut from fresh leaves supportedby plastic foam, using a sledge microtome. These were mounted in Honda medium and ex-amined in a Zeiss Photomicroscope III using Nomarski interference or phase-contrast optics.

Chloroplast number per cell, chloroplast area and cell area were measured in separated cellsof glutaraldehyde-fixed tissues, as previously described (Possingham & Smith, 1972) for5 experiments. The percentage of dumbbells per cell was also measured for one of theseexperiments.

Scanning electron microscopy. Disks were fixed in 3 % glutaraldehyde, o-i M sodium phosphatebuffer for 2-4 h at room temperature, washed in buffer and postfixed in aqueous 1 % osmiumtetroxide. Additional osmium was bound to the tissue using thiocarbohydrazide as a ligand(Kelley, Dekker & Bluemink, 1973). After being dehydrated through an acetone series, thespecimens were dried from liquid CO8 in a Balzers Union Critical Point Dryer, and fragmentedby cutting or tearing. The tissue pieces were mounted on stubs and examined in a Philips500 SEM. Some samples were gold-coated in an ISI PS-2 sputter coater before observation.

Freeze-etching. Cultured leaf disks and the bases of leaves less than 2 cm in length, fresh orfixed in 3 % glutaraldehyde, o-1 M sodium phosphate buffer, were processed in a Balzersfreeze-etch unit. Fixed tissue was transferred to 25 % glycerol for a minimum of 4 h beforefreezing in 25 % glycerol on gold specimen disks in liquid Freon 22 at —150 °C.

Fresh tissue was surrounded by water and frozen as above. Freeze-etch replicas were cleanedby an initial soaking in 80 % sulphuric acid overnight at room temperature followed by 4-5 hat 60 °C. After being rinsed in water, the replicas were transferred to sodium hypochlorite(3'5 % available chlorine) for 4 h, rinsed in distilled water and mounted.

RESULTS

Cell size, chloroplast number and percent dumbbells

When leaf disks cultured for 5 days in low-intensity green light were exposed to high-intensity white light, the area of their cells increased by 80% over 16 h (Fig. 1).Changes also occurred in the number and conformation of the chloroplasts (Fig. 1).After a lag period of about 8 h, the number of chloroplasts per cell increased by nearly65 % over the next 5 h. Furthermore, at t0, approximately 65 % of the chloroplastswere in a dumbbell configuration, whereas after 16 h in white light, only 20% of thechloroplasts were constricted. At f16, the unconstricted, ovoid chloroplasts frequentlyoccurred in mirror-image pairs.

Chloroplast division in spinach leaves 89

Light microscopy

Sections of living tissue were cut from disks cultured for 5 days in green light andexamined using Nomarski interference optics. Only cells exhibiting protoplasmicstreaming were observed and photographed (Figs. 2, 3). The cells contained chloro-plasts which were either approximately round or dumbbell-shaped, i.e. oblong and

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Fig. i. Changes in cell area (# , bars indicate S.E., 5 experiments), chloroplast numberper cell (A, bars indicate S.E., 5 experiments and percentages dumbbells per cell ( • ,bars indicate S.E. except where ±0-05, one experiment) in disks grown for 5 days ingreen light and then transferred to white light.

constricted to varying degrees near the centre (Fig. 2). Some chloroplasts were in themobile or amoeboid phase (Fasse-Fransisket, 1956). These chloroplasts were irregularin outline; pseudopodia-like projections were common and, in some instances,measured several microns in length. Occasionally, 2 chloroplasts appeared to be con-encted by such projections (arrow, Fig. 3), but careful examination disclosed a smallgap between them. The grana in these chloroplasts were not always evenly distributed,

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N. Chaly, J. V. Possingham and W. W. Thomson

Chloroplast division in spinach haves 91

as areas free of grana were often observed at the periphery of the chloroplasts and atthe extremities of the dumbbell-shaped organelles (Fig. 2).

Small structures similar in appearance to mitochondria, were also numerous inliving cells. They frequently lay clustered near the chloroplasts.

SEM

Chloroplasts were located in a single layer close to the cell wall (Fig. 4). They wereshaped either like flattened spheroids 1-1-15 ^m m diameter or like dumbbells (Figs.5, 6) measuring 1-1-5 Z4111 by I'5~2/'m> a nd were about 0-2-0-3 /im deep. Dumbbell-shaped chloroplasts could be differentiated from pairs of unconnected ovoid chloro-plasts (Fig. 4). Although chloroplasts were constricted to varying extents, in mostcases they were in an hourglass configuration (Fig. 5). Some chloroplasts showed adistinct neck of variable diameter connecting the 2 halves of the dumbbell (Fig. 6).

Organelles similar in shape and size to mitochondria were frequently observedadjacent to chloroplasts (Fig. 4). In some instances, they appeared to lie in the grooveformed by the constriction in a dumbbell.

Freeze-fracture and freeze-etch

Chloroplast shape varied considerably. Chloroplast profiles were circular, lenticularor oblong in shape (Figs. 7-16). Some oblong chloroplasts showed only a slight in-dentation near the middle (Fig. 10). Others were more markedly pinched in at thecentre, forming a dumbbell-shape. Asymmetrical constrictions were observed occasion-ally. Whereas in most instances chloroplasts were the shape of an hourglass (Figs.11, 12, 15), occasionally a tube-like bridge of uniform diameter was seen between the2 postulated daughter chloroplasts (Figs. 13,14). Occasionally, dumbbell-shaped chloro-

Figs. 2, 3. Nomarski interference-contrast microscopy of live cells in spinach leaves.

Fig. 2. The cell contains many dumbbell-shaped chloroplasts of varied size. Granularregions in the centre of the organelle are generally surrounded by a homogeneous cleararea. The outline of many chloroplasts is irregular, and projections of diverse size andshape can be seen (arrows). Many small structures (arrowheads), probably mito-chondria, are visible near the chloroplasts. x 1160.

Fig. 3. Only a narrow gap (arrow) remains between pseudopodium-like projectionsof adjacent chloroplasts, creating a superficial image of a single, dumbell-shapedchloroplast. x 1630.Figs. 4-6. Scanning electron micrographs of fixed leaf disks.

Fig. 4. Cell in which numerous mitochondria-like bodies (small arrowheads) appearto be associated with the chloroplasts (large arrowheads, constricted chloroplasts).x 8340.

Fig. 5. Portion of cell containing dumbbell in an hourglass shape, x 17150.

Fig. 6. Constricted chloroplast in which the two halves are joined by a tube-likebridge (cf. Fig. 13). x 8900.

Fig. 7. Hourglass-shaped chloroplast in a freeze-etch preparation of an unfixedleaf disk, x 10200.

Fig. 8. Portion of constricted chloroplast in a freeze-etch preparation of unfixed youngleaf base tissue, x 23800.

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N. Chaly, J. V. Possingham and W. W. Thomson

Figs. 9-16. Freeze-etching of fixed leaf disks.Fig. 9. Part of cell showing the envelope surface of 3 chloroplasts (2 dumbbells,

1 ovoid) and the stroma and thylakoids (arrow) of a fourth, x 14800.Fig. 10. Oblong chloroplast with a slight indentation near the centre.The unevenness

of the surface suggests an irregularity of outline as described in Fig. 2. x n 600.Fig. 11. Hourglass-shaped chloroplast clearly showing the continuity of the surface

membranes from one half to the other, x 16400.

Chloroplast division in spinach leaves 93

plasts with surface irregularities similar to small pseudopodia-like projections seen inliving cells (Figs. 2, 3) were observed in the freeze-etch preparations (Fig. 14).

The planes of fracture occurred so as to show the envelope (Figs. 7, 9-11, 13, 14)or the stroma (Figs. 7, 9, 12, 15, 16). The chloroplasts were most commonly fracturedin a plane perpendicular to that of the thylakoid sheets (Figs. 7, 15, 16); occasionally,however, the fracture exposed large areas of thylakoid membrane as it passed throughthe organelle in the same plane as the thylakoids (Fig. 12).

The system of thylakoids appeared continuous throughout the length of thedumbbell-shaped profiles. Membranes frequently lay within the narrowed portionof the dumbbell (Figs. 12, 15) and at no point during division were 2 'daughter'systems evident.

Dumbbell-shaped chloroplasts were also observed in replicas of unfixed leaf disks(Fig. 7) and of unfixed young leaves (Fig. 8).

DISCUSSION

Despite the lack of direct evidence, such as has been provided by cinematographyfor lower plants (Green, 1964), good indirect evidence is now available that there isa precursor—product relationship between dumbbell-shaped chloroplasts and newchloroplasts. In the present experiment with leaf disks, the number of chloroplastsper cell increased by nearly 65% within 12 h. Over the same period, the proportionof dumbbell-shaped chloroplasts decreased by 65%. It could perhaps be argued thatthe dumbbell represents a change in chloroplast shape induced by green light andthat it is not strictly a division figure. The present results suggest that this is unlikely,as a correlation exists between the timing and extent of the increase in plastid numberand the decrease in the percentage of dumbbells. Furthermore, electron-microscopeobservation of these disks provides no evidence of proplastids, etioplasts or otherplastid forms that might represent an alternative pathway for chloroplast formation.

The large size of the chloroplast makes it almost impossible to focus clearly on theentire organelle in the light microscope, particularly in the area of the constriction.With SEM or with transmission electron microscopy of freeze-etch replicas it ispossible, however, to distinguish between constricting chloroplasts and shapes thatform when chloroplasts overlap or emit pseudopodia-like structures. In the presentwork, we have demonstrated that chloroplasts with dumbbell configurations exist in2 tissues where chloroplast numbers are known to be increasing rapidly, i.e. culturedspinach leaf disks and young spinach leaves. Many constricted chloroplasts wereobserved in SEM preparations of fixed material and in freeze-etch replicas of fixedand unfixed leaf and leaf disk tissue. Taken in conjunction with the data presented inFigs. 1-3, we believe these observations demonstrate unmistakably that dumbbell-shaped chloroplasts exist in vivo and represent a stage in the division of chloroplasts.It is interesting to note at this stage the considerable similarity in the conformation ofisolated and intracellularly located dumbbells, even to the presence of pseudopodia-like projections, when observed by Nomarski interference-contrast microscopy, SEMand freeze-etching.

94 N. Chaly, J. V. Posstngham and W. W. Thomson

Chloroplast division in spinach leaves 95

We have observed dumbbells constricted to various degrees, suggesting a divisionsequence from round plastid to elongated cylinder to slightly indented cylinder, andthen passing either directly to an almost complete constriction or, more rarely,passing through a stage with a tube-like connection between the 2 daughter plastids.Somewhat similar constriction figures have been observed previously with the lightmicroscope (Ridley & Leech, 1970; Possingham, 1976; Vujicic & Possingham,unpublished; see also Kameya & Takahashi, 1971).

The mechanism by which the constriction develops is unknown. As in maturechloroplasts of Agapanthus (Fasse-Fransisket, 1956), the constriction was generallycentrally located. Both SEM and freeze-etch preparations suggest that the constrictionencircles the chloroplast, as though formed by the tightening of a drawstring. Thefreeze-etch planes of fracture lay parallel or perpendicular to the thylakoid sheets.Dumbbell-shaped chloroplasts with both orientations of thylakoids were observed,suggesting that the constriction occurs both in the breadth and the thickness of theorganelle, i.e. encircles the chloroplast. No structures either inside the plastid, suchas microtubules, or outside in the cytoplasm, such as attachment fibres, could beresponsible for generating the constriction.

We thank M. Buttrose, P. Scholefield and M. Sedgley for advice on SEM and freeze-etching.We also thank Miss P. Cain, Mr E. Lavvton, Mrs R. Luters and Mr A. Soeffky for technicalassistance. Professor W. W. Thomson was in receipt of a Fulbright grant from the AustralianAmerican Education Foundation during his period in Australia.

REFERENCES

BOASSON, R. & LAETSCH, W. M. (1969). Chloroplast replication and growth in tobacco. Science,N. Y. 166, 749-751.

BOFFEY, S. A., ELLIS, S. G., SELLDEN, G. & LEECH, R. M. (1979). Chloroplast division andDNA synthesis in light-grown wheat leaves. PI. Physiol., Lancaster 64, 502-505.

CRAN, D. G. & POSSINGHAM, J. V. (1972). Two forms of division profile in spinach chloroplasts.Nature, New Biol. 235, 142.

ESAU, K. (1972). Apparent temporary chloroplast fusions in leaf cells of Mimosa pudica. Z.PflPhysiol. 67, 244-254.

FASSE-FRANSISKET, U. (1956). Die Teilung der Proplastiden und Chloroplasten bei Agapanthusuvibellatus l'Herit. Protoplasma 45, 194-227.

Fig. 12. Hourglass-shaped chloroplast fractured in the plane of the thylakoids (t).These lie throughout the organelle, even in the area of the constriction, x 17200.Fig. 13. Constricted chloroplast with tube-like bridge connecting the 2 sides. A mito-chondrion (m) lies in the cytoplasm between the neck of the dumbbell and anotherchloroplast (c). x 19 200.Fig. 14. Constricted chloroplast as in Figure 13, but with a pseudopodium-like projec-tion (p) at one end. x 13000.Fig. 15. Hourglass-shaped chloroplast fractured in a plane perpendicular to that of thethylakoids (arrows). These extend longitudinally throughout the profile, and are notinterrupted in the constricted area, x 14400.Fig. 16. Constricted chloroplast with a central tube-like bridge. The plane of fracturehas disclosed the stroma and thylakoids in part of the organelle, showing that theyextend longitudinally, x 21 200.

96 N. Chaly, J. V. Possingham and W. W. Thomson

GREEN, P. B. (1964). Cinematographic observations on the growth and division of chloroplastsin Nitella. Am. J. Bot. 51, 334-342.

KAMEYA, T. & TAKAHASHI, N. (1971). Division of chloroplast in vitro. Jap. J. Genet. 46, 153-157-

KELLEY, R. O., DEKKER, R. A. F. & BLUEMINK, J. G. (1973). Ligand-mediated osmium bind-ing: its application in coating biological specimens for scanning electron microscopy.J. Ultrastruct. Res. 45, 254-258.

KIRK, J. T. O. & TILNEY-BASSETT, R. A. (1978). The Plastids. Amsterdam: Elsevier/North-Holland Biomedical Press.

LANCE, A. (1958). Infrastructure des cellules du meristeme apical et des jeunes ebauchesfoliaires de Chrysanthemum segetum L. (Composers). j4miis.Sct.nat.,Bot. 1 ieserie, 19,165-202.

LEECH, R.( RUMSBY, M. G. & THOMSON, W. W. (1973). Plastid differentiation, acyl lipid andfatty acid changes in developing green maize leaves. PI. Physiol., Lancaster 52, 240-245.

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POSSINGHAM, J. V. (1980). Plastid replication and development in the life cycle of higher plants.A. Rev. PI. Physiol. (In Press.)

POSSINGHAM, J. V. & SAURER, W. (1969). Changes in chloroplast number per cell during leafdevelopment in spinach. Planta 86, 186-194.

POSSINGHAM, J. V. & SMITH, J. W. (1972). Factors affecting chloroplast replication in spinach.J. exp. Bot. 23, 1050-1059.

RIDLEY, S. M. & LEECH, R. M. (1970). Division of chloroplasts in an artificial environment.Nature, Lond. 227, 463-465.

SCHIMPER, A. F. W. (1885). Untersuchungen tiber die Chlorophyllk6rper und die ihnenhomologen Gebilde. Jb. toiss. Bot. 16, 1-247.

SENSER, F. & SCHOTZ, F. (1964). Untersuchungen iiber die Chloroplastenentwicklung beiOenothera. II. Der Albivelutina-typ. Planta 62, 171-190.

WHATLEY, J. M. (1974). Chloroplast development in primary leaves of Phaseolus vulgaris.Neta Phytol. 73, 1097-1110.

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{Received 27 May 1980)