Ultrastructural changes of chloroplasts and mitochondria have been observed in synchronously growing cells of Euglena gracilis Z, under photoautotrophic conditions. Application of the serial section technique allows estimation of the number and volume of these organelles. Several 3-dimensional reconstructions reveal their shape and distribution throughout the cell cycle.

In young cells 10 separate diskoid or branched chloroplasts are found. They show the typical lamellar structure of euglenoid chloroplasts. During the growth phase (light period), they enlarge and their volume doubles. Some of them branch out, so that 20 lobes are formed. Thylakoids grow longer without change in number. The pyrenoid persists only during the first half of this period. During the cell division phase (dark period), branched chloroplasts divide along 2 planes which are perpendicular to each other and perpendicular to the thylakoid plane. All thylakoids are cut and their number does not change in the daughter chloroplasts. The plastidome volume constitutes 15–18% of the total cell volume over the entire life cycle.

One of the most significant observations in this report is the presence of a single permanent mitochondrial reticulum during the whole cell cycle. This giant mitochondrion consists of an extremely branched network with delicate threads (0·4–0·6 μm thick) surrounding the chloro-plasts, nucleus and reservoir. It extends throughout the cell. During the growth phase, it becomes gradually longer and doubles in volume. The degree of branching increases but the thickness of the threads remains constant. During the division phase, the mitochondrial elements appear more restricted (0 ×4 μm thick) and the reticulum becomes progressively partitioned into 2 daughter networks. At any time of the cell cycle, the chondriome volume is about 6% of the total cell volume.

These results are discussed in comparison with numerous relevant papers on fight and electron microscopy of animal and plant cells. They suggest that the descriptions of several authors on the plastidial cycle and the mitochondrial cycle in Euglena, both said to be characterized by alternate reticulate and fragmentary states, arise in part from questionable interpretation of random sections. It is evident that the form and distribution of organelles can be determined more precisely by serial sectioning.

The great plasticity of chloroplasts and mitochondria in Euglena cells, in response to various culturing conditions, delayed, until recently, detailed studies on the behaviour of these organelles during the life cycle. The recent research has been intimately correlated with the perfecting of culture media and obtaining synchronously grown cells, and its main result has been to characterize a reticular and a fragmentary phase of plastidome and chondriome during one cell cycle (Calvayrac, 1972; Michaels & Gibor, 1973; Lefort-Tran, 1974; Osafune, Mihara, Hase & Ohkuro, 1975 a, b, c; Calvayrac & Lefort-Tran, 1976). In our opinion, tridimensional reconstructions of 8 variously branched mitochondria based upon about 64 ultrathin serial cell sections (Osafune et al. 1975c) and one of a branched, irregularly shaped chloroplast, built from a few semithin serial sections (Calvayrac & Lefort-Tran, 1976), seemed in-adequate to prove the transitory presence of a reticular phase of these 2 organelles in non-dividing cells and we had even stronger reservations about the proposed existence of a fragmentary mitochondrial phase. This latter interpretation disregarded the fact, previously reported by Leedale & Buetow (1970) and Moore, Cantor, Sheeler & Kahn (1970) and clearly demonstrated with 3-dimensional models of the chondriome in Chlorella (Atkinson, John & Gunning, 1974) and Euglena (Osafune et al. 1975c), that small ovoid structures, obviously separate on micrographs, might form part of branched elements and even of a single reticulum.

In the present study, we have tried to provide some indisputable proof of reticulate or particulate phases of chloroplasts and mitochondria by ultrathin serial sectioning of entire cells of Euglena gracilis, these cells being harvested throughout the growth and division periods of synchronous cultures. Moreover, this technique has also allowed study of quantitative changes of chloroplasts and mitochondria during the life cycle.

All cells of Euglena gracilis Z were obtained from the algal culture collection of the University of Gdttingen (strain no. 1224 5/25).

Synchronous cultures

Euglena cultures were made in 1-l. flasks containing 500 ml of the inorganic salt medium (pH 6·8) described by Cramer & Myers (1952) and modified by Padilla & James (1960). Algal cell suspensions were aerated with filtered air enriched with CO, (5%) at 22 °C. Cell synchronization was obtained by photoperiods under a 14-h light-10-h dark regime. Illumination was about 10000 lux. The cell number was kept constant (about 105 cells per ml) during 15 generations by diluting by half with fresh medium at the beginning of each light period. With the same culturing conditions, Schantz (1972) distinguished 2 clearly distinct phases in the cell cycle of green Euglena by studying changes in cell number, average cell volume, amounts of dry material, total nitrogen and chlorophyll. The growth phase coincided with the light period whereas cell division occurred during the dark period.

Electron microscopy

Glutaraldehyde was added to a cell suspension in culture medium to give a final concentration of 1% which was left standing for 10 min at room temperature. The cells were harvested by gentle centrifugation and fixed in a newly made fixative solution (5% glutaraldehyde in 01 M phosphate buffer, pH 7· 4) for 2 h at room temperature. They were rinsed in three 10-min changes of the same buffer and postfixed for 1 h in 2% osmium tetroxide in acetate— veronal buffer (pH 7 ·4). After being washed rapidly in the same buffer, they were embedded in 2% agar which was cut into 1-mm’ blocks. These blocks were dehydrated in a graded ethanol series, embedded in a mixture of Epon and Araldite and sectioned on a Porter Blum MT2 ultramicrotome with a diamond knife. Ribbons were about 60–80 serial sections of about 100 ran thickness. These sections were transferred in pairs to narrow single-slit copper grids coated with Formvar. An entire cell was included in about 150 serial sections.

The sections were stained with uranyl acetate and lead citrate according to Reynolds (1963). The periodic acid-thiocarbohydrazide-silver proteinate method of Thiery (1967) was sometimes used to obtain a better contrast of cytomembranes (25 min oxidation in 1% aqueous periodic acid, 2·5 h in thiocarbohydrazide, 30 min in silver proteinate in darkness). All sections were examined using a Philips EM-300 electron microscope.

Three-dimensional reconstructions

Three-dimensional models of several cells showed the shape, area and distribution of nucleus, chloroplasts, mitochondrion and reservoir. They were made by tracing the outlines of organelle profiles from micrographs of ultrathin serial sections onto thin plywood which equalled in thickness the thickness of ultrathin sections (0·1 μm) multiplied by the magnification of the negative. All fragments were numbered and, after cutting, stacked up.

Determination of organelle numbers and volumes

All sections of one cell were photographed and then traced from micrographs. The outlines of cell profiles were drawn tangentially to the external ridge of the pellicle striations. Those of chloroplasts, mitochondrion, nucleus and reservoir sections were also drawn. By superimposing the tracings of a single cell, it was possible to follow the continuity of organelles and to reveal discontinuities, and thus to estimate the number of each type of organelle. The area of cell and organelle profiles was calculated by polar planimetry. As the average thickness of sections was known, it was easy to calculate the volume of the cell and that of every organelle. However, being conscious of the limitations of this method, we have provided all results (in μm2) in whole numbers except for some percentages which have one place of decimals. Indeed, decimal and values to several places, often reported in papers, are not justifiable in relation to the lack of precision in the methods used, so they seem to us unnecessary.

These results concern electron-microscope observations of 8 cells which have been respectively chosen (a) during the light period, at the first hour (young cells nos. 1 and 2), at the seventh hour (cell no. 3) and at the thirteenth hour (aged cell no. 4), and (1) during the dark period, at the fourth hour (cells at metaphase nos. 5 and 6) and at the sixth hour (cell at telophase no. 7 and 2 daughter cells nos. 8 A and 8B at late cell cleavage).

Quantitative results are recorded in Tables 1 to 3.

Table 1.

Quantitative changes during a cell cycle

Quantitative changes during a cell cycle
Quantitative changes during a cell cycle

Plastidome

Morphology and fine structure

Chloroplast sections of photoautotrophically grown Euglena gracilis often appear elongated or spindleshaped (Fig. 1), or more rarely branched as a complex lobed structure (Fig. 2). Generally, each contains 5 to 8 lamellae composed of 2–3 closely appressed thylakoids in both growing and dividing cells. During the first half of the light period, a differentiated region can be discerned in most chloroplast sections. This is the pyrenoid, much denser and more finely granular than the surrounding chloroplast matrix, traversed by some lamellae and often bounded by extraplastidial paramylon (Fig. 3).

Figs. 1, 2.

Median sections of cells showing spindleshaped chloroplasts, a complex lobed one (C) and numerous mitochondrial profiles (mr). ca, canal; cv, contractile vacuole; N, nucleus; r, reservoir. Thiéry test. Fig. 1, × 9000; Fig. 2, × 10200.

Figs. 1, 2.

Median sections of cells showing spindleshaped chloroplasts, a complex lobed one (C) and numerous mitochondrial profiles (mr). ca, canal; cv, contractile vacuole; N, nucleus; r, reservoir. Thiéry test. Fig. 1, × 9000; Fig. 2, × 10200.

Fig. 3.

Portion of a non-dividing cell containing a chloroplast (C) with a pyrenoid (py) surrounded by paramylon (pa), d, dictyosome; rnn, nuclear membrane; mr, mitochondrial reticulum; N, nucleus; pe, pellicle. Stain, potassium permanganate, × 18500.

Fig. 3.

Portion of a non-dividing cell containing a chloroplast (C) with a pyrenoid (py) surrounded by paramylon (pa), d, dictyosome; rnn, nuclear membrane; mr, mitochondrial reticulum; N, nucleus; pe, pellicle. Stain, potassium permanganate, × 18500.

Fig. 4.

Tangential section of a non-dividing cell enclosing.an elongated and branched mitochondrial profile (mr). Stain, potassium permanganate, × 14600.

Fig. 4.

Tangential section of a non-dividing cell enclosing.an elongated and branched mitochondrial profile (mr). Stain, potassium permanganate, × 14600.

Distribution

Examination of serial sections has revealed that chloroplasts are separate organelles during the entire cell cycle. They are regularly located at the periphery of the cell, each with its long axis parallel to the cell surface (Figs. 5, 6). They lie with a minimum overlap and so get maximum light. Occasionally, some are scattered throughout the cytoplasm. Most cells possess both diskoidal or flattened chloroplasts and branched or deeply lobed chloroplasts (Figs. 711). The most branched one observed extended as a 5-lobed structure (Figs. 12, 13). However, some cells (for example cells nos. 1 and 7) contain only lens-shaped chloroplasts.

Every diskoidal chloroplast includes 1 pyrenoid or, more rarely, 2, one of the two lying in a central location. In branched chloroplasts, each lobe encloses a pyrenoid.

Figs. 5, 6.

Disposition of chloroplasts in cell No. 3 (Fig. 5) collected at the seventh hour of the non-division phase, and in cell no. 4 (Fig. 6) harvested at the thirteenth hour of the same period, ca, canal; N, nucleus; r, reservoir. Fig. 5, × 3400. Fig. 6, ×3000.

Figs. 5, 6.

Disposition of chloroplasts in cell No. 3 (Fig. 5) collected at the seventh hour of the non-division phase, and in cell no. 4 (Fig. 6) harvested at the thirteenth hour of the same period, ca, canal; N, nucleus; r, reservoir. Fig. 5, × 3400. Fig. 6, ×3000.

Figs. 7–11.

Different 3-dimensional reconstructions of chloroplasts. Fig. 7. Diskoidal chloroplast, × 5800.

Figs. 7–11.

Different 3-dimensional reconstructions of chloroplasts. Fig. 7. Diskoidal chloroplast, × 5800.

Fig. 8.

The constriction (arrow), perpendicular to the long cell axis divides the chloroplast into 2 end-to-end lobes, × 5800.

Fig. 8.

The constriction (arrow), perpendicular to the long cell axis divides the chloroplast into 2 end-to-end lobes, × 5800.

Figs. 9–11.

Growth of chloroplasts to form finally U-shaped organelles. The arrow’ on Fig. 10 points to the beginning of a constriction parallel to the long cell axis, × 4800.

Figs. 9–11.

Growth of chloroplasts to form finally U-shaped organelles. The arrow’ on Fig. 10 points to the beginning of a constriction parallel to the long cell axis, × 4800.

Figs. 12, 13.

Two opposite views of the complex-lobed chloroplast of young cell no. 2 (first hour of the non-division phase). There are 5 lobes (1 to 5). The thick arrows parallel to the long cell axis, show the X-shaped structure (lobes 1 and 2). The thin one, perpendicular to the long cell axis, indicates the narrow constriction joining the 2 lobes 1 and 3. × 10200.

Figs. 12, 13.

Two opposite views of the complex-lobed chloroplast of young cell no. 2 (first hour of the non-division phase). There are 5 lobes (1 to 5). The thick arrows parallel to the long cell axis, show the X-shaped structure (lobes 1 and 2). The thin one, perpendicular to the long cell axis, indicates the narrow constriction joining the 2 lobes 1 and 3. × 10200.

Quantitative changes during the cell cycle

The number of chloroplasts and plastidial lobes varies during the cell cycle (Table 1). Young cells contain about 10, there being respectively 8 and 7 in the 2 cells nos. 1 and 2 collected at the first hour of the cycle, and 7 and 9 in the daughter cells nos. 8 A and 8 B. During the first hour of a new cycle, at the beginning of the light period, every chloroplast or plastidial lobe contains one pyrenoid. During the first 7 h of the growth phase, the number of chloroplasts does not change but the plastidial lobes approximately double in number. In cell no. 3, fixed at the seventh hour of the cycle, chloroplasts are branched into 18 lobes with a pyrenoid in every one. During the second half of the light period (from the seventh to the fourteenth hour of the cycle), the number of chloroplasts and plastidial lobes remains constant but pyrenoids disappear. Thus 10 chloroplasts, branched into a total of 19 lobes, are found in the aged cell no. 4 at the thirteenth hour of the cycle, but no pyrenoids are observed in the lobes. This absence of pyrenoids has been confirmed by numerous observations of random ultrathin sections of cells harvested at the eighth, eleventh, twelfth and fourteenth hours of the cycle.

In all dividing cells (dark period), pyrenoids are lacking. During the first 5 h of that period, the branched chloroplasts divide into diskoidal elements so that their number per cell approximately doubles. Thus, there are 17 and 14 chloroplasts in cells nos. 5 and 6 at metaphase, 20 in cell no. 7 at late telophase and 16 in the 2 daughter cells nos. 8A and 8B. This division may involve all the chloroplasts, in consequence of which cells will contain only diskoidal organelles (for example, young cell no. 1 and cell no. 7 in telophase), or just some of them (for example, daughter cells nos. 8 A and 8B at late cleavage and young cell no. 2).

At cytokinesis, the distribution of chloroplasts may be variable. The 2 observed daughter cells contain 7 and 9 chloroplasts which are divided into 8 and 12 lobes respectively. Indeed, at the end of cell cleavage, some organelles may be confined within the cytoplasmic bridge joining the 2 daughter cells where they may undergo a constriction and then, possibly, division.

Plastidome volume

At any time of the cell cycle, the total chloroplast volume varies between 15 and 18% of the cell volume (Table 1). This small variation proves that there is a good correlation between growth of plastidome and that of the cell. However, the chloroplasts included in a single cell sometimes have different volumes (Table 2). The average volume is about 20 μm3 in young cells and 50 μm3 in aged cells. During the cell division phase, the separation of plastidial lobes brings down the average volume to 20 μm3.

Table 2.

Volume of chloroplasts (in μm 2)

Volume of chloroplasts (in μm 2)
Volume of chloroplasts (in μm 2)

The total volume of the pyrenoids of one cell in relation to the chloroplast volume ranges from 5·2 to 7·2% (Table 3). These values indicate clearly that the pyrenoid volume increases rapidly during the first hour of the light period.

Table 3.

Volumes of chloroplasts (C) and pyrenoids (in μm3). Percentages of pyrenoid volume in comparison with chloroplast volume

Volumes of chloroplasts (C) and pyrenoids (in μm3). Percentages of pyrenoid volume in comparison with chloroplast volume
Volumes of chloroplasts (C) and pyrenoids (in μm3). Percentages of pyrenoid volume in comparison with chloroplast volume

The percentage equivalent to 4·7, calculated for cell no. 3 at the seventh hour of the cycle, in which the plastidial volume has doubled, indicates good correlation between the increase of chloroplast volume and that of pyrenoid volume from the first to the seventh hour of the light period. After this, the pyrenoids disappear as rapidly as they appeared.

Chondriome

Morphology and distribution

Mitochondrial profiles in photoautotrophic cells generally appear in the micrographs as small ovoids (0·4 × 0·6 μm), though elongated (Figs, 1, 2) or branched structures are occasionally seen, the latter usually in tangential cell sections (Fig. 4).

By serial sectioning, it is easy to prove that most profiles, observed separately in individual sections of a cell, really represent parts of an extensive network, though a few of them may belong to one, or more rarely 2 or 4, small mitochondrial fragments.

Three-dimensional models show the distribution of the mitochondrial reticulum and clearly prove the existence of a single giant mitochondrion which is variously branched but present in all cells over the entire life cycle (Figs. 1421). This reticulum extends uniformly throughout the cell. It consists of some elements confined between the plasmalemma and the external surface of the chloroplasts and others situated more deeply in the cytoplasm. It constitutes a large-mesh network, closely apposed against chloroplasts, nucleus and reservoir, with some branches having independent extremities.

Figs. 14, 15.

Three-dimensional reconstructions of cell no. 1 (first hour of the nondividing phase). Two opposite views. The cell encloses 8 simple chloroplasts (C) (each with a pyrenoid) and a mitochondrial reticulum (mr) with large meshes, ca, canal; cv, contractile vacuole; N, nucleus; r, reservoir, ×6000.

Figs. 14, 15.

Three-dimensional reconstructions of cell no. 1 (first hour of the nondividing phase). Two opposite views. The cell encloses 8 simple chloroplasts (C) (each with a pyrenoid) and a mitochondrial reticulum (mr) with large meshes, ca, canal; cv, contractile vacuole; N, nucleus; r, reservoir, ×6000.

Morphological changes during the cell cycle

The mitochondrial reticulum undergoes morphological and quantitative changes during the entire cell cycle. Its flattened elements in young cells are about 0·4–0·6 μm thick (Figs. 14, 15, 18). During the growth phase, in the light period, the reticulum undergoes branching but the threads remain slightly flattened in transverse section. In dividing cells, during the dark period, elements become thinner along their whole length and these delicate threads acquire a circular diameter of about 0·4 μm. This aspect persists during the entire cell division period. The 3-dimensional model of a cell at metaphase (Figs. 16, 17, 19) and that of a cell at late cleavage (Figs. 20, 21) reveals that the chondriome is formed of more delicate tubular elements than those observed on the model of a young cell (Figs. 14, 15, 18). At the beginning of the division phase, the ramifications passing through the plane of cell division stretch and divide. In the cell at late cytokinesis, chondriome division is complete (Figs. 20, 21, arrows point to elements with an independent extremity situated between the 2 daughter cells). Therefore, even before their separation, the newly formed daughter cells possess a single mitochondrial reticulum each.

Figs. 16, 17.

Three-dimensional reconstructions of cell no. 5 at metaphase (fourth hour of the division phase). Two opposite views. The cell contains 17 chloroplasts (C) branched into 20 lobes (fragmentation phase of chloroplasts). The mitochondrial reticulum (mr) is formed by numerous delicate threads 0·4 μm thick, cv, contractile vacuole; N, nucleus; r, reservoir, ×5200.

Figs. 16, 17.

Three-dimensional reconstructions of cell no. 5 at metaphase (fourth hour of the division phase). Two opposite views. The cell contains 17 chloroplasts (C) branched into 20 lobes (fragmentation phase of chloroplasts). The mitochondrial reticulum (mr) is formed by numerous delicate threads 0·4 μm thick, cv, contractile vacuole; N, nucleus; r, reservoir, ×5200.

Figs. 18, 19.

Views of the single mitochondrial reticulum in young cell no. 1 (Fig. 18, × 5700), and in cell no. 5 at metaphase (Fig. 19, × 5400). The reticulum becomes more complex in aged cells.

Figs. 18, 19.

Views of the single mitochondrial reticulum in young cell no. 1 (Fig. 18, × 5700), and in cell no. 5 at metaphase (Fig. 19, × 5400). The reticulum becomes more complex in aged cells.

Figs. 20, 21.

Three-dimensional reconstructions of daughter cells nos. 8A and 8B harvested at the sixth hour of the division phase (cytokinesis). Two opposite views. The dumb-bell shape results from the euglenoid movements of the cell. Each daughter cell contains several chloroplasts (C) and a single mitochondrial reticulum (mr). The arrows point to the breakage of mitochondrial threads at the cytoplasmic bridge. N, nucleus; r, reservoir, ×4800.

Figs. 20, 21.

Three-dimensional reconstructions of daughter cells nos. 8A and 8B harvested at the sixth hour of the division phase (cytokinesis). Two opposite views. The dumb-bell shape results from the euglenoid movements of the cell. Each daughter cell contains several chloroplasts (C) and a single mitochondrial reticulum (mr). The arrows point to the breakage of mitochondrial threads at the cytoplasmic bridge. N, nucleus; r, reservoir, ×4800.

Chondriome volume

The chondriome volume estimated as a percentage of the total cell volume varies only between 4·7 and 5-7 during the cell cycle (Table 1). These values indicate parallel rates of cytoplasmic and mitochondrial synthesis. During the growth phase, the chondriome volume doubles and then remains constant during the division period. At cytokinesis, each daughter cell receives approximately half of the mother cell chondriome. For example, the chondriomes of daughter cells nos. 8A and 8B total 61 and 63 /tm3 respectively.

The regular intracellular distribution and nearly constant thickness of the mitochondrial elements in growing cells, together with the doubling in chondriome volume, imply 2-fold extension of the total length of the mitochondrial reticulum.

Plastidome

Number of chloroplasts. The presence of 8–10 diskoidal or elongated chloroplasts has been reported in photoautotrophically-grown cells of Euglena gracilis Z by some authors (Gojdics, 1953; Schiff, Lyman & Epstein, 1961; Schiff, 1971, 1973; Schantz, 1972; Cook, 1973; Cook & Li, 1973; Cook, Haggard & Harris, 1976) or 3-5 large chloroplasts, with the same culturing conditions, by others (Lefort-Tran, 1974; Orcival-Lafont & Calvayrac, 1974; Calvayrac & Lefort-Tran, 1976).

It may be asked whether these differences of opinion arise from the chosen strain, from the growth conditions used prior to experimentation, or from the various analytical techniques. The present study, based upon serial sections through entire cells, clearly proves the existence of about 10 chloroplasts.

Modes of chloroplast division

Numerous papers have described chloroplast division but our observations provide further details about the direction of the division plane and the point of division in the cell cycle.

Von Wettstein (1954) described 2 possible methods of chloroplast division in the phaeophyte Fucus vesiculosas. Division could be parallel or perpendicular to the lamellae. In the first case, the daughter chloroplasts received only a portion of the lamellae and lamellar replication was necessary to restore the basic number. On the other hand, Cook et al. (1976) reported the presence of 6-8 lamellae in sections of the chloroplasts of both young and aged cells of Euglena gracilis and so ruled out the possibility of a division parallel to the thylakoids.

We agree with this latter view

The number of lamellae appears constant (6-8) at any time of the cell cycle. Three-dimensional reconstructions of chloroplasts of all cells collected throughout an entire cycle demonstrate that the division plane always cuts all thylakoids perpendicularly. Dividing diskoidal chloroplasts show one or two major or minor constrictions which are perpendicular to the long cell axis and produce 2 or 3 lobes (Fig. 8). This division has often been reported, for example in the chrysophyte Ochromonas danica (Gibbs, Cheng & Slankis, 1974), in the chiorophyte Derbesia tenuissima (Wheeler & Page, 1974) and in the rhodophyte Odonthalia floccosa (Goff, 1976). When the chloroplast of Euglena bends and becomes U-shaped during its development, some constrictions may extend into the curvature so that the plastidial profile takes on a Y- or X-shape. Such division pictures have been described in Euglena by Schiff & Epstein (1968), Schiff (1971) and Schantz (1972). According to Von Wettstein (1954), they would form from divisions parallel to the thylakoids in Fucus. Our models demonstrate (Figs. 913) that they arise from constrictions which cut all thylakoids and make their way towards the long cell axis. So the 2 types of constrictions observed (i.e. perpendicular and parallel to the long cell axis) expand in 2 planes which are perpendicular to each other and perpendicular to the thylakoid plane (Fig. 22). Simultaneous progress along these division planes allows the development of complex branched chloroplasts such as the five-lobed structure of young cell no. 2 (Figs. 12, 13).

Fig. 22.

Schematic drawing of a dividing chloroplast. The division planes P, and Pt are perpendicular to each other and perpendicular to the lamellar plane. They cut all thvlakoids and partition the chloroplast into lobes (Z and L). The arrows indicate: the direction of progress of constriction in planes P1-P′, (thick arrows), in plane Ps (thin arrows) and the direction of chloroplast extension (great arrows). Plane P1 is parallel to plane P1. Plane P, indicates the orientation of the ultrathin sections.

Fig. 22.

Schematic drawing of a dividing chloroplast. The division planes P, and Pt are perpendicular to each other and perpendicular to the lamellar plane. They cut all thvlakoids and partition the chloroplast into lobes (Z and L). The arrows indicate: the direction of progress of constriction in planes P1-P′, (thick arrows), in plane Ps (thin arrows) and the direction of chloroplast extension (great arrows). Plane P1 is parallel to plane P1. Plane P, indicates the orientation of the ultrathin sections.

Period of chloroplast division

The position of chloroplast division during the cell cycle of Euglena gracilis has been discussed in several papers. Chloroplasts have been said to divide before cell division (Petropoulos, 1964; Cook, 1966; Orcival-Lafont, Pineau, Ledoigt & Calvayrac, 1972; Lefort-Tran, 1974; Ettl, 1976) or else during mitosis (Leedale, 1967; Schiff, 1971; Cook, 1973; Boasson & Gibbs, 1973; Richard & Manning, 1974). These differences of opinion perhaps arise in part from a staggered distribution of chloroplast divisions within a single cell. Hence, to provide some valuable information about the phase of these divisions, it is necessary to determine, as Boasson & Gibbs (1973) did, if they are better synchronized within one cell than in the whole culture. The latter authors inferred, after observation of frequency histograms of chloroplast numbers per cell during a cell cycle in synchronous culture, that chloroplast division occupied from 6 to 8 h and occurred during the normal 10-12 h of cell division. However, they concluded that the chloroplasts of an individual cell did not divide in strict synchrony with each other. Our results also prove that there is synchrony between chloroplast and cell divisions. The number of chloroplasts doubles during the first 6 h of the cell division period, but the presence of regularly shaped chloroplasts in young cell no. 1 and cell no. 7 at late telophase and of branched chloroplasts in young cells nos. 2, 8 A and 8B provides evidence of imperfect synchrony of division within individual cells.

Morphological changes during the cell cycle

The mode of chloroplast division has been reported time after time, but the morphological development of these organelles during a cell cycle has been described less often. In Euglena gracilis, Orcival-Lafont et al. (1972) and Calvayrac & Lefort-Tran (1976) determined, after a stereological study and one 3-dimensional reconstruction of a massive irregularly shaped chloroplast from a few serial semithin sections, that a chloroplast reticulate phase occurred during the cell growth period and a phase in which chloroplasts divided into fragments occurred immediately before cell division. It is evident that a few serial sections may reveal that organelle profiles, apparently separate in an individual section can be interconnected, yet it seems inadequate information from which to infer that a branched chloroplast is part of a single plastidial reticulum. Our results do not confirm the existence of such a reticulum at any time of the cell cycle. Chloroplasts of Euglenagracilis, with our culturing conditions, are always separate structures although some are occasionally very branched ones. Their morphological development throughout a cell cycle comprises 2 clearly distinct phases: an extending and branching phase during the cell growth period and a dividing phase of plastidial lobes during cell partition. It is therefore possible to consider the notion of the chloroplast division period during the cell cycle differently according to whether one identifies this division from the formation of plastidial lobes (before cell division) or from their separation (during mitosis).

In addition to these cyclic branching and dividing activities of the chloroplasts, there are cyclic changes in the distortion and inflation of chloroplasts and also in the appearance of pyrenoids. Cook & Li (1973) and Cook et al. (1976) described, in photoautotrophically grown cells of Euglena gracilis, contorted chloroplasts with closely apposed lamellae during the light period and swollen chloroplasts with widely separated lamellae during the dark period. They considered these morphological modifications in relation to cyclic changes of the internal pH of cells. Cyclic changes in lamellar location and dispersal of chloroplast matrix have not been detected in our experiments.

Development of pyrenoids

Pyrenoid development during the cell cycle has been reviewed by Brown, Arnott, Bisalputra & Hoffman (1967), Griffiths (1970), Dodge (1973) and Bisalputra (1974). According to numerous authors, especially Bourquin (1917), Hartmann (1921), Czurda (1928), Simon (1954), Manton & Leedale (1961), Murakami, Morimura & Takamiya (1963), Brown & Bold (1964), Gantt & Conti (1965), Manton (1966) and Leedale (1967) pyrenoids divide before chloroplast division but other authors, for example, Smith (1914), Czurda (1928), Szejnman (1933), Giraud (1962), Bisalputra & Weier (1964), Brown & Bold (1964), Kônitz (1965), Arnott & Brown (1966), Hoffman (1968) and Atkinson et al. (1974) describe pyrenoid disappearance before or during cell partition and formation de novo in the chloroplasts of the daughter cells. On the other hand, Cook et al. (1976) assumed a cyclic development of pyrenoids in Euglena gracilis, after examination of numerous random micrographs. They suggested that the pyrenoid appeared at the beginning of the light period and disappeared in the middle of that period (though they cautiously pointed out that the pyrenoid occupies only a small region inside the chloroplast and that most random sections might not show this structure).

The present study agrees with Cook et al. (1976). Pyrenoids are always present in cells collected during the first 7 h of the light period but are lacking in aged cell no. 4 harvested at the thirteenth hour of the light period, and also in all cells selected during the dark period. Moreover we have never observed pyrenoids in numerous random micrographs of cells fixed at the eighth, eleventh, twelfth and fourteenth hours of the light period. By serial sectioning, it is easy to determine the location and area of pyrenoids in every chloroplast. The percentage of sections of a single cell without pyrenoid but with nuclear profile fluctuates between 15 and 20%. It follows that the conclusions of Cook et al. (1976), founded on the examination of several hundred micrographs, seem to us convincing evidence for the cyclic development of pyrenoids in photoautotrophically grown Euglena.

Quantitative changes of the plastidome

Several authors have reported data about the number of chloroplast sections per cell and about the average length and breadth of chloroplast profiles but only a few of them have estimated the volume of these organelles.

In Euglena gracilis Z growing in a medium with lactate as sole carbon source for growth, Orcival-Lafont et al. (1972) showed by the stereological method that the average volume of a chloroplast in cells harvested just before the division period varied between 242 and 343 μm3. Such cells contained about 8 chloroplasts (Orcival-Lafont & Calvayrac, 1974). It is thus possible to infer that the average volume of the plastidome in daughter cells would change from 968 to 1372 μm3. These values are markedly higher than those reported in the present study (Table 1) and perhaps result from different culturing conditions. It is unfortunate that the authors did not specify the percentage of plastidome volume in relation to cell volume. This percentage might also be higher than ours (15·8 to 18·3%) since otherwise the cells would reach a volume between 5200 and 8600 /im3; conversely, in a cell of about 3000 μm3, the plastidome volume would be between 32 and 45% of the cell volume. Although similar values have been found by Schdtz, Bathelt, Arnold & Schimmer (1972) in Chlamydomonas reinhardii and by Atkinson et al. (1974) in Chlor ella fusca vacuolata, they seem to us unlikely in Euglena. Indeed, cells growing under constant light in photoautotrophy or in photoheterotrophy (Pellegrini, 1978a), contain only 5 to 6 large chloroplasts and the plastidome volume is always about 16% of the cell volume.

Chondriome

Mitochondrial reticulum

The great plasticity of mitochondria is a unanimously accepted phenomenon since its demonstration by Lewis & Lewis (1914). However, the number, shape and development of these organelles during the cell cycle still remain much debated topics in both animal and plant cells.

The presence of a filamentous and very extensive chondriome was reported, for the first time, by Volkonsky (1930) without him realizing it. He described, in the protist Polytoma uvella, a reticulate ‘leucoplast’ which was, in fact, as suggested by Pringsheim (1948), the chondriome. Similarly, according to A. Hollande (1952), the mitochondrial nature of the internal filamentous network in dinoflagellates was not recognized by Chatton (1933) or Hovasse (1933) who identified it as a leucoplast. Chadefaud (1932) was the first to describe a reticulate chondriome in Chlorophyceae. Such branched mitochondria, extending throughout the cell, were afterwards observed by light microscopy in dinoflagellates (Biecheler, 1934), Volvocales (Hovasse, 1937), cryptomonads and euglenoids (Hollande, 1940, 1942a, b). Then Buvat (1946a, b, Mftfa, b, 1948a, b, 1953) discovered hypertrophied mitochondria in higher plants, especially in living root cells of Cichorium intybus. These chondriomes were from too to 150 μm long, often longer than the cells themselves. He also referred to the presence of giant mitochondria in senile cells and in cells in which cyclosis had been stopped by various processes.

In 1948, while continuing Hollande’s researches on euglenoids, Hovasse (in Pringsheim & Hovasse, 1948) applied the thin serial section technique, recommended by him as early as 1937. He easily identified the chondriome and was astonished at the total failure of his predecessors to do so. He characterized the mitochondrial network in photoautotrophic Euglena with outstanding exactness. That description, written 30 years ago, can be applied to the mitochondrial network of our 3-dimensional models, and it is with admiration and due homage to the author that we quote it here: ‘C’est un réseau typique formé de filaments grêles et calibrés, dessinant des mailles irrégulières dans toute la cellule, et autour des chloroplastes. Ce réseau présente de nombreuses extrémités libres, qui peuvent se résoudre en chondriocontes, ou même en mitochondries (s.s.).’

The first profiles of mitochondria observed in thin sections with the electron micro-scope seemed to cause the reticular structure of the chondriome of some cells and its great plasticity to be forgotten. Indeed, Wolken & Palade (1953) in their ultrastructural study of two flagellates, especially Euglena gracilis, and Wolken (1967) recalled the researches of Hovasse but did not compare their electron micrographs with observations obtained with the light microscope. Until recently, the works of Hovasse have not often been referred to in the numerous papers concerning the chondriome and it became established that the mitochondrion was ovoid or spherical but liable to hypertrophy under various experimental conditions. It was only in 1970, after the discovery of a mitochondrial network in Euglena spirogyra by Leedale, Meeuse & Pringsheim (1965) and Leedale (1966) that Leedale & Buetow (1970) re-established the important concept of the mitochondrial network, by correlated light- and electron-microscope observations. They observed, using phase-contrast light microscopy, the unceasing motions of fragmentation and fusion of the filamentous mitochondrial threads in living streptomycin-bleached cells of Euglena gracilis var. bacillaris. They found that the chondriome was always a reticulum, either delicate and complex in control cells, or thick and more restricted in carbon-starved cells. The authors recalled the papers of Lewis & Lewis (1914) and Frederic (1958) concerning the great plasticity of the chondriome and were surprised that the moving reticulum might be interpreted as spherical and motionless organelles by some cytologists. They wrote, ‘Despite the elegant demonstratison of the lability of mitochondria in many living cells, the study of mitochondrial structure by electron microscopy has tended to produce an impression of immobile rod-shaped organelles. Indeed, ovoid profiles are often described as “mitochondria” without the understanding that they may represent sections of a single complex reticulum.’ According to these authors, the separate ovoid mitochondrial profiles reported in particular by De Haller (1959), Gibbs (1960), Siegesmund, Rosen & Gawlik (1962), Malkoff & Buetow (1964) and Leedale (1967) all represented sections of a reticulum and not ovoid mitochondria. Moore et al. (1970) likewise suggested that the number of mitochondria in the flagellate Poly-tomella agilis, estimated from random sections, was overvalued in comparison to that found with the serial section method.

In spite of these counsels of prudence, papers still describe the presence or absence of a mitochondrial reticulum on the basis of suitable micrographs which show either elongated and branched structures or else ovoid profiles. Such interpretations have been put forward by Calvayrac (1972), Calvayrac, Butow & Lefort-Tran (1972), Ledoigt, Calvayrac, Orcival-Lafont & Pineau (1972), Michaels & Gibor (1973), Calvayrac, Bertaux, Lefort-Tran & Valencia (1974) and Osafune et al. (1975 a, b, c) who described a fragmentary phase and a reticular phase of the chondriome of Euglena gracilis. Yet, earlier and more convincing techniques, based upon serial sectioning, were available to show the spatial configurations of cells or organelles. Indeed, in a review of these methods, Ware & Lo Presti (1974) recalled that the first spatial reconstruction was built by His as early as 1880. The application of these techniques to the analysis of ultrathin serial sections, evolved by Sjostrand (1958, 1967), later allowed the number, shape, volume and spatial relations of various cell organelles to be established either by schematic or graphical representations or by 3-dimensional reconstructions. So, over the last 10 years, numerous authors have provided irrefutable proof of the reticular nature of the chondriome in various types of cell, for example, in the spermatium of the hemipteran Murgantia histrionica (Pratt, 1968), in the yeast Saccharomyces cerevisiae (Hoffmann & Avers, 1973; Stevens, 1974), in the chiorophytes Chlorella fusca vacuolata (Atkinson et al. 1974) and Chlamydomonas reinhardii (Grobe & Arnold, 1975, 1977), in the apoplastidial flagellate Polytomella agilis (Burton & Moore, 1974), in the leucoplastidial flagellate Polytoma papillatum (Gaffai & Kreutzer, 1977), in the kinetoplastid flagellates Blastocrithidia culicis and Trypanosoma cruzi (Paulin, 1975), Cryptobia vaginalis (Vickerman, 1977), Bodo sp. (Brugerolle & Mignot, 1979) and in the cryptophyte Cryptomonas sp. (Santore & Greenwood, 1977), while Osafune et al. (1975c) described, in 2 aged cells of Euglena gracilis a branched mitochondrial structure from 38 and 64 serial sections and Rohr (1978) assumed its existence in Ginkgo biloba with suitable sections.

Our results are founded on more complete series of sections (about 150 sections per cell) and we have already reported the presence of a single permanent mitochondrial reticulum which consists of thin delicate threads in the photoautotrophically grown cells of Euglena gracilis or a fenestrated peripheral shell-like structure in etiolated cells in darkness, intermediates forms being visible during greening and de-greening of cells (Pellegrini, 1976, 1978 a, b\ Pellegrini & Pellegrini, 1976; Schantz & Pellegrini, 1976-77).

Role of the mitochondrial reticulum or isolated mitochondria

According to Hobbs (1971) the elongated mitochondria of the volvocalean Eudorina illinoiensis might agglomerate in cell regions of high oxidative activity. On the other hand, Osafune, Mihara, Hase & Ohkuro (1972) and Osafune et al. (1975a) noted a parallel development between the decrease of oxygen-uptake activities and formation of giant mito-chondria in Chlamydomonas reinhardii and Euglena gracilis. According to Heywood (1977), the chondriome aspect might depend upon the cell volume. The author compared his results with those obtained by Manton (1959) on the flagellate Micro-monas pusilia, Manton & Parke (1960) on another flagellate Micromonas squamata, Keddie & Barajas (1969) on the yeast Pityrosporum orbiculare, Schôtz et al. (1972) and Grobe & Arnold (1975) on Chlamydomonas reinhardii, Grimes et al. (1974) and Hoffman & Avers (1973) on the haploid and diploid cells of the yeast Saccharomyces cerevisiae, Atkinson et al. (1974) on Chlorella fusca and Rancourt, McKee & Pollack (1975) on the lymphocytes of mouse. He noted that in the 2 chloromonadophycean algae Gonyostomum semen and Vacuolaria virescens, there were either filamentous mitochondrial elements or else a single mitochondrial reticulum in the small cells (< 300 μ m3) whereas the larger cells (> 4000 μ m3) contained numerous mitochondria. He suggested that permanent fragmentation of the chondriome might allow a more homogenous distribution of mitochondria in the large cells.

Our observations do not agree with this view. In small cells of Euglena (< 1000 μ m3) as in the larger ones (> 3000 μ m3), the chondriome is always netlike. Moreover, our models demonstrate an extensive repartition of the mitochondrial threads, and the endless fragmentation and fusion of which, observed in living Euglena by Leedale & Buetow (1970), satisfy the requirements of various cell regions as efficiently as would separate mitochondria.

Morphological changes during the cell cycle

An alternative between reticular and granular mitochondrial phases has been described during the cell cycle in photo-autotrophically-grown cells of Euglena gracilis by Ledoigt et al. (1972), Calvayrac (1972), Osafune (1973), Calvayrac et al. (1974), Lefort-Tran (1974) and Osafune et al. (1975a, b). We do not agree with this interpretation for the authors described these 2 states after single observation of random sections even though, in 1970, Leedale & Buetow wrote that the presence of separate small mitochondrial profiles is not alone enough to prove the inexistence of a reticulum. We think so. Indeed the models of one or several filamentous mitochondrial elements in Chlamydomonas reinhardii (Schdtz, 1972; Arnold, Schimmer, Schdtz & Bathelt, 1972; Schdtz & Bathelt, 1972; Schdtz et al. 1972; Grobe & Arnold, 1975), in Chlorella fusca vacuolata (Atkinson et al. 1974), in Euglena gracilis (Osafune et al. 1975c), in lymphocytes of mouse (Rancourt et al. 1975) and in ascites tumour cells (Koulk, Vorbeck & Martin, 1977) clearly showed that all cell profiles (related to serial sections) contained small mito-chondrial elements. While making the first serial sections of Euglena, Osafune et al. (1975c) described, from 64 serial sections, 8 very branched filamentous mitochondria present just before the cell division period and ruled out the possibility of a fragmentation into many independent small mitochondria.

In fact, our reconstructions of a permanent mitochondrial reticulum seem completely convincing since they are built from the observation of serial sections through several entire cells harvested at different times of the cell cycle. This exclusive reticulum was found, even when, in our material, cell profiles contained only small mitochondrial elements.

This single, permanent, variously branched mitochondrion, observed in living Euglena gracilis var. bacillaris by Leedale & Buetow (1970) and proved here in Euglena gracilis, is not a unique structure. Such a chondriome has been described in Saccharo-myces cerevisiae by Hoffman & Avers (1973) and in Chlorella fusca vacuolata by Atkinson et al. (1974). Without wanting to make too great a generalization of this phenomenon, we nevertheless think that the presence of a reduced number of giant mitochondria already described in several cell types indicates the possible existence of a mitochondrial reticulum in which the various branches take shape, grow longer, stretch themselves, part, retract and coalesce constantly without the intervention of cyclic fragmentation processes. The presence of either numerous branched mito-chondria or a single mitochondrial reticulum might be explained by different frequencies according to species and growth conditions, of the number of sites where fragmentation and fusion of mitochondrial threads occur. The frequency might be reduced in Euglena gracilis where the chondriome is always a single reticulum but might be greater in lymphocytes of mouse which contain 2 or 3 large mitochondria (Rancourt et al. 1975) and still greater in Chlamydomonas reinhardii which has between 9 and 14 branched mitochondria (Schdtz, 1972; Arnold et al. 1972; Schdtz et al. 1972; Grobe & Arnold, 1975).

Quantitative volumetric changes. The mitochondrial volume has not often been published. It ranges from about 2 to 20% of the cell volume in Chlamydomonas reinhardii (Schdtz et al. 1972), Chlorella fusca vacuolata (Atkinson et al. 1974), Saccharomyces cerevisiae (Stevens, 1977) and Polytomella agilis (Burton & Moore, 1974) and varies just a little (about 1%) during the cell cycle of one species. In Euglena gracilis, growing on lactate medium, the average mitochondrial surface, calculated for too /zm2 of the cell surface by the stereological method (Calvayrac et al. 1974) reduces slightly (24-9 to 22-6 μm2) during the non-division phase and undergoes sudden and important augmentation (16-7 to 24-9 μm2) at the end of cytokinesis. The authors infer augmentation of the mitochondrial volume in relation to cell volume between the division phase and the non-division phase of the cell but do not provide volumetric values. On the other hand, ultrathin serial sectioning allowed to us evaluation of the mitochondrial volume for each cell (Table 1) and showed in Euglena gracilis growing on the inorganic salt medium of Cramer & Myers (1952) that the percentage of the mitochondrial volume in comparison to cell volume varies just a little (4-7 to 5’7%) during the cell cycle. Although our culturing conditions were different from those described by Calvayrac et al. (1974), augmentation of the chondriome volume pointed by these authors at the end of cytokinesis seems to us surprising.

Despite the tempting explanation of plastidial and mitochondrial cycles by the alteration of a reticular phase and a fragmentary one, our results demonstrate that there is neither a plastidial reticular phase during the non-dividing cell period, nor a mitochondrial fragmentary phase during mitosis. However, these 2 organelles undergo parallel development over the cell cycle. Indeed, both the plastidial and mitochondrial volumes increase during the cell growth phase and remain constant during cell division. During the latter period, plastidial lobes and the mitochondrial reticulum divide and each daughter cell receives approximately half of the plastidial and mitochondrial volumes. The constant percentage of the volume of these organelles in relation to the cell volume suggests an effective regulation of cytological balance. Different morphological aspects of chloroplasts, shown on the 3-dimensional models, allow determination in time and space of 2 methods of plastidial division and understanding of the complex X- or Y-shapes of dividing chloroplasts, confused until now with a mode of constriction parallel to the lamellae.

Our results demonstrate the advantages of the serial section technique over the more usually applied stereological method. We have been able to calculate unambiguously the number of organelles per cell and to analyse their relative volumetric status. Moreover, serial sectioning provides the necessary elements for building 3-dimensional models. These models show the morphology, volume and distribution of organelles within the cell and consequently reveal their exact relationships. The possibility of coupling this method with the synchronous culture technique also avoids the disadvantages of making deductions from a comparatively small number of cells.

I am greatly indebted to Professor R. Buvat (Institut de Cytologie et de Biologie Cellulaire, Université Aix-Marseille II) for his continued interest and encouragement throughout this investigation and to Professor G. F. Leedale (Department of Plant Sciences, Leeds University, G.B.) for his helpful comments and suggestions on this work and for critically reading the manuscript. I am also grateful to Michel Berthoumieux and Roger Guerrini for their excellent technical assistance.

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