Planta 151,215 225 (1981)

9 Springer-Verlag 1981

Evolution of the Cytoplasmic Organdies During Female Meiosis in Pisum sativum L.* F.J. Medina, M.C. Risuefio and M.I. Rodriguez-Garcia** Morfoghnesis Celular, Instituto de Biologia Celular (C.S.I.C.), Velfizquez, 144, Madrid-6, Spain

Abstract. In this paper we have traced the evolution of the cytoplasmic organdies in the female germinal cell of P i s u m s a t i v u m L., from the beginning of meiosis to the early stages of the maturing megaspore, in order to correlate the morphological changes with the physiological aspects of megasporogenesis. A process of intense cytoplasmic vacuolation takes place in the megaspore mother cell ( M M C ) during prophase I, probably proceeding from the smooth endoplasmic reticulum and dictyosomes; it results in the formation of big vacuoles, which play a role in M M C polarization. By means of this polarization most plastids and mitochondria are incorporated into the functional megaspore at the end of meiosis. There are plastid and mitochondria cycles which consist of dedifferentiation followed by redifferentiation. During these cycles a transient morphology appears, called a cup-shaped form, which we interpret as an expression of low organelle activity. The wall of the M M C thickens throughout megasporogenesis and loses its plasmodesmata during middle prophase I. The ribosome population is reduced during prophase I and then restored during the early stages of the megaspore maturing process, as shown by the quantitative study that we have carried out. The nucleolar cytoplasmic bodies play a part in this restoring process. These bodies have a special morphology and appear to be originated from the activity of the nucleolar organizing region (NOR) during nucleolar disorganization in prophase I,

* This work has been partially supported by the "Comisidn Asesora para la investigacidn Cientifica by T6cnica" Projects n~613/02 and 613/10 ** Present a&h'ess: Departamento de Bioquimica, Estacidn Ex-

perimentaI dei Zaidin (C.S.I.C.), Prof. Albareda 1, Granada, Spain Abbreviations." EDTA = ethylene-diamine-tetracetic acid ; ER = endoplasmic reticulum (SER: smooth ER); MMC=megaspore mother cell; NOR=nucleolar organizing region; RNP-ribonucleoproteins

We think that this cytoplasmic evolution is a response to nuclear genic recombination, in order to provide the most adequate expression of the zygote genome. Key words: Meiosis (organelles) meiosis).

Organelles (during


Plant gametogenesis is one of the cellular processes in which the cell structures undergo a large number of changes in a short time. This enables us to study the morphological and physiological aspects of the organelles and the structure-function relationship in them. Male gametogenesis has been more extensively studied than female gametogenesis (see Heslop-Harrison's review 1971), though in the fertilization process it is the female cytoplasm not the male which is the zygote cytoplasm. Earlier works on megasporogenesis at the electron microscopic level (Israel and Sagawa 1964, 1965; Steward and Gifford 1967; Godineau 1968; Woodcock and Bell 1968) were too general, thus failing to observe the details of the evolution of each organelle. A more recent and detailed study by Dickinson and Potter (1978) pointed out the existence of a similar behavior in both male and female cytoplasm. In the present paper we undertake a complete study of the evolution of the cytoplasm during megasporogenesis in Pisurn s a t i v u m . This process has its own identity, which causes it to have special importance in the plant life cycle. Material and Methods

Ovaries were used from very young Pisum sativum L. flowers grown in a greenhouse. Severaldays before the corolla opened, the flowers



F.J. Medina et al : Cytoplasmic Organelles During Plant Female Meiosis

were excised and the ovaries dissected. They were fixed in 3% glutaraldehyde, buffered with 0.025 M cycodylate at pH 7 for 2 h at room temperature, post-fixed in 1% OsO4 in the same buffer for 1 h at room temperature, and then dehydrated in an alcohol series and embedded in Epon 812. Ultrathin sections were stained with 1% uranyl acetate in absolute ethanol for 30 min at room temperature and then with lead citrate according to Venable and Goggeshall (1965)

Cytochemistry a) Ribonucleoproteins. Ultrathin sections of glutaraldehyde-fixed, Epon-embedded ovaries were stained by the uranyl acetate-EDTAlead citrate method, according to Bernhard (1969).

b) Silver Impregnation. (Risuefio et al. 1973). The ovaries were fixed overnight at 4 ~ C in a mixture of 10% formol and 1% hydroquinone. Silver impregnation with 2% AgNO3 was carried out at 7 0 ~ in the dark for 4 h. The material was then postfixed for one hour at room temperature in formol-hydroquinone. After usual embedding and ultramicrotomy, sections were observed directly, without staining, under the electron microscope.

Quantitative Study of the Ribosome Population. We have assumed the following : a) The organelle distribution (nucleus, vacuoles, ribosomes, plastids, mitochondria, etc.) in a central section is the most representative of the entire ceil; so, the data obtained from the surface of this section can be extended to cover the entire cell volume. b) the more central the section, the larger the cell surface which it contains. On central sections of each stage, the areas of the entire cell, nucleus, and vacuoles were measured. We also took several micrographs from various sections at each stage (8-15 per stage) and drew squares of 1 pm 2 on them over the cell surface and counted the number of ribosomes enclosed. We avoided the inclusions of vacuolar cytoplasm in the squares. For each stage we calculated the average number of ribosomes in all 1 p m 2 squares counted; this number was multiplied by the area in g m a of the germinal cell, excluding the areas of both, the nucleus and the vacuoles. The resulting number was divided by the entire area of the germinal cell, giving the total average number of ribosomes per g m z.

Vacuolation : Endoplasmic Reticulum (ER) and Golgi Apparatus. During middle prophase I, a vacuolation process takes place in the M M C in such a way that, in pachytene, a large part of the M M C volume becomes vacuolar cytoplasm (Fig. 1). In stages prior to the formation of big vacuoles, we have observed an increase in the amount of smooth endoplasmic reticulum (SER) (Fig. 4). At the same time, the dictyosomes, which in leptotene appear to be rather scarcely developed (Fig. 3), are seen during zygotene in a higher number and clearly show vesicles in their neighborhood which are without doubt the products of their activity (Fig. 4). Throughout the zygotene phase the number of small vesicles in the whole M M C gets progressively higher (Fig. 4). In pachytene, when the vacuolation process is just finished, the SER is still well developed, but no small vesicles are observed in the MMC, either beside the dictyosomes or dispersed. In this stage, dictyosomes again show a similar profile to that of leptotene, that is, with features corresponding to an inactive organelle (Fig. 2). In the dyad, there is a clear difference between the cytoplasm of both sister cells: The micropylar cell is mostly vacuolated and, as far as we can see, it appears practically devoid of cell organelles (Fig. 7); in contrast, the chalazal cell is mainly nonvacuolated and has cell organelles such as plastids (Fig. 8). Plastids and Mitochondria. In the first stages of meiosis - leptotene and almost all zygotene - the plastids and mitochondria of MMC are small in size, and their internal membrane system shows little development; both organelles have a "meristematic-like" morphology (Fig. 3).


In this work we have studied the female haploid phase of the reproducing cycle of Pisum sativum from the beginning of meiosis to the first steps of the functional megaspore maturing process. Early in this period, a considerable enlargement of the megaspore mother cell (MMC) occurs. After the first nuclear division, a cell wall is formed between both dyad nuclei. As a result of meiosis, four possible megaspores can be produced, but only one - the nearest to the ovular chalaza develops as a functional megaspore, while the other three degenerate. Immediately after finishing meiosis, the functional megaspore begins to mature by greatly enlarging itself. Embryogenesis of Pisum sativum is of the Polygonum type, i.e., monosporic, eight-nucleate embryo sac (Cooper 1938 ; Maheswari 1950).

Fig. 1. General survey of the M M C during the pachytene stage, the longest period of meiotic prophase I. The cytoplasm shows an intense vacuolation; two large vacuoles (v) are seen in this section. Most of the organelles are confined to the chalazal region (Ch) while the micropylar part (Mp) appears devoid of them giving the M M C a marked polarization. F r o m these organelles, cupshaped plastids are clearly recognizable (arrows). The nucleus (N) occupies a slightly micropylar position. Bar = 3 ~tm, x 3,900 Fig. 2. Magnification of a portion of the M M C during pachytene, showing cup-shaped plastids (P) with the stroma denser than the matrix of mitochondria (34). The latter are extremely elongated and have cristae only in the wider spaces of the organelle. Endoplasmic reticulum cisterns (ER) are abundant. Dictyosomes (d) are small and show no signs of vesicle-emitting activity. A nucleolar cytoplasmic body is present (ncb) with grey matrix and very dense granules. N, Nucleus; v, vacuole; cw, cell wall. B a r = l gin, x 26,700

F.J. Medina et al: Cytoplasmic Organeiles During Plant Female Meiosis



F.J. Medina et al: Cytoplasmic Organelles During Plant Female Meiosis

Fig. 3. Cytoplasmic organelles in the MMC during leptotene. Plastids (P) and mitochondria (34) are small and have a "meristematiclike" morphology with few internal lamellae. Dictyosomes (d) are scarcely developed. Note the high ribosome density of the cytoplasm. Bar= t pm, x 31,500 Fig. 4. Cup-shaped plastids (P) in the MMC cytoplasm during late zygotene. We can see diverse morphological profiles depending on the section planes. Endoplasmic reticulum cisterns (ER) are near them. Dictyosomes (d) are active in emitting vesicles (arrowheads). Several kinds of small vesicles can be observed (arrows) either in the neighbourhood of dictyosomes or dispersed. Bar= 1 pm, x 51,000

F.J. Medina et al: Cytoplasmic Organelles During Plant Female Meiosis

CZ3 h p........ ~



Fig. 5. Schematic drawing of the MMC during pachytene showing the polarity in the distribution of the organelles. A, micropylar part; B, central part; C, chalazal part; N, nucleus; V, vacuoles; P, plastids; M, mitochondria From late zygotene on, the plastids become cupshaped. This is an important transformation that consists of a lengthening of the plastids, a differential thickening of some parts, and a folding over (Figs. 1, 2 and 4). Their stroma is dense and granular with few internal lamellae and little clear spaces. Frequently, endoplasmic reticulum cisterns are near them (Fig. 4). The cup-shaped plastids are present in M M C during late zygotene and the whole pachytene stage. Mitochondria are transformed in a similar way to plastids, but the transformation begins in pachytene, that is, later than in the case of plastids. Cristae are only located in the wider spaces of the cup-shaped mitochondria, while the narrow spaces are almost tubular, without structures between the surrounding double membranes (Fig. 2). The matrix density of cup-shaped mitochondria is lower than the stroma density of plastids (Fig. 2). In diplotene, neither typical nor cup-shaped forms of platids and mitochondria are present, but only undifferentiated, double membrane-bound organelles with very electron transparent matrices or stromas, their internal lamellae system being scarcely developed. In their vicinity, large quantities of smooth endoplasmic reticulum are located (Fig. 6). After the end of meiosis, when the megaspore


maturing process begins, the plastids initiate a progressive redifferentiation prior to mitochondria. The internal lamellae system of the plastids develops slowly, and it becomes more and more frequent to find starch grains in them (Fig. 9). This redifferentiation process passes through a cup-shaped phase (Fig. 9). As well as in the late zygotcne stage, endoplasmic reticulum cisterns can be observed in the megaspore very near to the plastid envelopes (Fig. 9).

The MMC Polarization in Prophase L F r o m the time at which the vacuolation process takes place in late zygotene, a marked polarization of the M M C occurs which then remains till the end of prophase I. This polarization is due to a shift of the nucleus to a position that is slightly micropylar, and also to the presence of most of the plastids and mitochondria at the chalazal pole (Fig. 1). If we consider a section of the whole M M C and divide this cell into three equal parts transversely to the micropyle-chalaza axis, we will find that the part nearest to the micropyle contains a relatively large vacuole and almost all the nucleus; the central part is occupied by a big vacuole, and the part nearest to the chalaza contains most of the plastids and mitochondria (Fig. 5). The Cell Wall. During the first stages of meiotic prophase I, the MMC wall does not appear to be different from the walls of the other ovular cells. It is a primary wall similar to that of a meristematic cell, 0.07-0.13 gm thick with plasmodesmata present in it (Fig. 10). Throughout prophase I, a slight thickening of the cell wall occurs, and the plasmodesmata disappear completely. At the end of meiosis, in maturing megaspore, the structure of the wall corresponds to a secondary wall, without plasmodesmata, and has a width of approximately 0.3 gm (Fig. 11). The Ribosome Population and the Cytoplasmic Bodies of Nuclear Origin. From the quantitative study that we have performed on the evolution of the ribosome population, we have observed a reduction in the number of ribosomes per surface unit in the course of meiosis. This reduction is followed by an increase to the original number when the megaspore maturing process begins (Fig. 15). During this evolution of the ribosome population, some bodies appear in the MMC cytoplasm. They consist of a matrix of medium electron density with several strongly osmiophilic granules immersed in it (Figs. 2, 12). These bodies were 0.3 lain in diameter and the osmiophilic granules, 50 nm. They were present in the M M C from late zygotene to the end of prophase I. Their location is variable but they are frequently found in the neighborhood of the nucleus.


F.J. Medina et al: Cytoplasmic Organelles During Plant Female Meiosis

F.J. Medina et al: Cytoplasmic Organelles During Plant Female Meiosis


Figs. 10 and 11. The cell wall (cw) at the beginning of meiosis (Fig. 10) and in maturing megaspore (Fig. 11). Differences in thickness and structure are evident. Fig. 10 shows a primary wall with plasmodesmata (pc/) and Fig. 11 shows a secondary wall fully devoid of plasmodesmata MMC: megaspore mother cell. Mg, megaspore; Nc, nucellus cells. Fig. I0 Bar= 1 pro, 39,000; Fig. 11 Bar= I pm, x 14,700 Figs. 12, 13 and 14. Nucleolar cytoplasmic bodies (ncb). With conventional fixation and staining techniques (Fig. 12) they show a grey fibrous matrix with some osmiophilic granules immersed in it. The same structure is seen using the EDTA technique which is preferential for R N P (Fig. 13). Employing the silver impregnation technique that is preferential for nucleolar material they appear strongly impregnated although their structure cannot be differentiated due to the resolution level of the technique. N: nucleus, Nu: nucleolus, cyt: cytoplasm. Fig. 12 Bar= 1 gm, x 46,300; Fig. 13 Bar= 1 pro, x 30,800; Fig. 14 Bar= 1 ~m, x 15,400

Fig. 6. M M C cytoplasm in diplotene. Both, plastids and mitochondria (arrows) show a marked dedifferentiation, with their matrix or stroma very electron-transparent and few internal membranes. Cup-shaped profiles are not observed. Endoplasmic reticulum cisterns (ER) are abundant and located among the organelles. Bar = 1 gm, x 23,200 Figs. 7 and 8. Two serial sections of the dyad under the light microscope. A cell wall (cu,) is present between both cells. Fig. 7 shows the micropylar dyad ceil (MD) with large vacuoles (v) in the cytoplasm. Fig. 8 mainly shows the chalazal dyad cell (ChD), which is less vacuolated. Cell organelles (probably plastids) can be seen in this cell (arrow). Fig. 7 B a r = 5 pm, x2,500; Fig. 8 B a r = 5 gm, x 2,200 Fig. 9. Maturing megaspore. Redifferentiating plastids (P) with a relatively dense stroma and starch grains (s). Cup-shaped profiies are present (arrow). Note their proximity to endoplasmic reticulum cisterns (arrowhead). Bar = 1 pro, x 33,900


F.J. Medina etaL : Cytoplasmic Organelles During Plant Female Meiosis

In some cases they possess an internal clear space (Fig. 12). The EDTA technique, which preferentially stains RNP, shows that their structure is the same as that seen with glutaraldehyde-osmium fixation (compare Figs. 12 and 13). The silver impregnation technique, preferential for nucleolar material, reveals cytoplasmic bodies positively impregnated (Fig. 14). For this reason we have named them nucteolar cytoplasmic bodies. Table l, summarizes the main results of the evolution of cytoplasmic organelles as reported above.


2_74 255



:~ 200,

196 183


no doto available


trom diplotene till


early maturing me~x~re ~)3..~o~ Fig, 15. Diagram of the quantitative study of the evolution of the ribosome population. Abscissa: developmental stage of megasporogenesis. Ordinate: average number of ribosomes per gm 2 in all micrographs counted belonging to one stage


Cell Vacuolation" Endoplasmic Retieulum and Golgi Apparatus. Role in M M C Polarization. Some authors have reported the presence of vacuoles during plant

Table 1. Summary of the main events that occur in the evolution of cytoplasmic estructures during megasporogenesis.


Zygotene Early

Cell Wall



Rest of Meiosis

Maturing Early

Megaspore Later



no plasmodesmata


Decreasing (elimination)

increasing (repopulation) )

I, . . . .




Nucleolar cytoplasmic bodies

Bodies of Nuclear Origin

Vacuoles in micropylar dyad cell


Abundant; small; difficult to distinguish from plastids

Evolution of the cytoplasmic organelles during female meiosis in Pisum sativum L.

In this paper we have traced the evolution of the cytoplasmic organelles in the female germinal cell of Pisum sativum L., from the beginning of meiosi...
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