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The nucleolar organizer (NOR) and fibrillar centers during plant gametogenesis
JOURNAL OF ULTRASTRUCTURERESEARCH 85, 3 0 0 - 3 1 0
(1983)
The Nucleolar Organizer (NOR) and Fibrillar Centers during Plant Gametogenesis F. J. MEDINA, 1 M. C. RISUENO, M. I. RODRIGUEZ-GARCIA, 2 AND M. A. SANCHEz-PINA Morfogdnesis Celular, Instituto de Biologfa Celular, CSIC, Velgtzquez 144, Madrid-6, Spain Received April 27, 1983 The ultrastructure and morphometric features of nucleolar fibrillar centers have been studied in three different plant species over a long period of gametogenesis, extending from premeiosis to after the first postmeiotic mitosis, with respect to variations in nucleolar activity which occur throughout this process. Fibrillar centers represent the structural counterpart of the transcriptionally inactive portions of the nucleolar organizing region (NOR); in some gametogenic stages, no discrete fibrillar centers can be observed and, therefore, the structure of the whole NOR has been studied. Two clearly differentiated types of fibrillar center structure are described, with a close relationship to different stages of nucleolar activity. Stages with high activity show "homogeneous" fibrillar centers, whose cbromatin is decondensed, and stages with low activity show "heterogeneous" fibrillar centers with both condensed and decondensed chromatin. These two types had previously been characterized cytochemically by us in somatic cells. Medium-active stages show fibrillar centers "intermediate" between the two main types. The morphometric behavior of fibrillar centers is also related to activity. The number of fibrillar centers per nucleolar section increases with the nucleolar activity, while their size decreases. However, the location of fibrillar centers in the nucleolus does not follow any special pattern, except in middle meiotic prophase I, in which the whole NOR occupies a peripheral position. This is due to a special NOR activity at these stages, whose products are stored as "nueleolar dense bodies." All these data allow us to conclude that fibrillar centers are highly dynamic structures, extremely sensitive to changes in nucleolar activity.
In a previous paper (Risuefio et al., 1982) we have shown that, in plant meristematic cells, the nucleolar fibrillar centers are made up of DNA and proteins, this DNA being inactive in transcription. The transcriptionally active nucleolar DNA is found in the dense fibrillar component. Both fibrillar centers and dense fibrillar component constitute a functional unit (Moreno Diaz de la Espina, 1976), being the structural counterpart of the nucleolar organizing region (NOR). So, the plant cell fibrillar centers are functionally identical to the animal cell ones (Goessens and Lepoint, 1979; Fakan and Puvion, 1980; Mirre and Stahl, 1981). However, the fibrillar centers of plant meristematic cells undergo changes that depend on the nucleolar activity. These changes afTo whom correspondence should be addressed. 2 Present address: Estaci6n Experimental del Zaidin (CSIC), Profesor Albareda, 1, Granada, Spain.
fect the ultrastructure and the morphometric features (number and size) of fibrillar centers. From a morphometric point of view, fibrillar centers are small and numerous in highly active nucleoli and larger and fewer as nucleolar activity drops (Medina et al., 1983). Ultrastructurally, in experimental conditions of reduced or suspended nucleolar activity, inclusions of condensed chromatin are seen in fibrillar centers, which are not present when nucleolar activity is high. This fact defines two types of fibrillar centers in plant cells, correlated with nucleolar activity: homogeneous, resembling very much those described in animal cells, and heterogeneous, which, in addition, contain condensed chromatin inclusions (Risuefio et al., 1982). This latter type is not usual in animal cells; it has only been reported in Nereis ovocytes (Bertout, 1983). Its existence in plants is most probably due to the number of ribosomal cistrons, much higher 300
0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
THE NOR DURING PLANT GAMETOGENESIS in plant than in animal cells (Ingle and Sinclair, 1972). In order to demonstrate whether or not this behavior o f fibrillar centers is general in plant cells we have l o o k e d for a plant cellular system that is completely different from the root meristem, and which has strong variations in its nucleolar activity under natural conditions. The system we have chosen is that o f reproducing tissues t h r o u g h o u t gametogenesis. During this process, the germinal cells pass, in a short time, through successive periods o f increased and decreased activity. The structure and activity o f the nucleolar organizer have been studied in a few stages o f plant male gametogenesis (Williams et aL, 1973; Esponda and G i m r n e z - M a r t l n , 1974, 1975; J o r d a n and Luck, 1976; Gim6nez-Martin et aL, 1977), but it has not been possible to reach any general conclusions on the structure-function relationship from these studies. For this reason, we have chosen a long period o f plant gametogenesis, extending from premeiosis up to the stages that c o m e after the first postmeiotic mitosis in both the male and female reproducing cells. During this period, the fibrillar centers undergo strong morphologic changes which we have related to the very different levels o f nucleolar activity that exist from one stage to another. Furthermore, the use o f three different species enables us to obtain conclusions o f b r o a d application for plant cells. MATERIALS AND METHODS Ovaries from Pisum sativum L. and anthers from Allium cepa and Hyacinthoides non-scriptus, all belonging to flowers grown in a greenhouse, were dissected from flower buds. For checking the stage of development, one anther from each flower was squashed in 2% acetic orcein. In the case of the ovaries, checking was only possible on semithin sections of fixed and embedded samples. Anthers were fixed with 3% glutaraldehyde in 0.05 Mcacodylate buffer for 2 hr at room temperature. The fixation of ovaries was carried out in the same way, except that the molarity of the buffer was 0.025 M cacodylate. Both anthers and ovaries were postfixed with 1%
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OsO4, in the same buffer that was used for glutaraldehyde fixation, for 1 hr at room temperature, then dehydrated in ethanol and embedded in Epon 812. Semithin sections were examined under the light microscope prior to ultramicrotomy. Ultrathin sections were cut on an LKB Ultrotome III, stained with uranyl acetate and lead citrate, and then observed under a Philips 300 electron microscope. Morphometry of Random Sections
We have calculated the number of fibrillar centers per section and their cross-sectional areas in those gametogenic stages displaying discrete fibrillar centers. Measurements of areas were carried out with a Kentron MOP/AM03 semiautomatic image analyzer. For both features in every stage, mean and standard deviation values were calculated and graphically represented. RESULTS Just before the beginning o f meiosis, the newly differentiated spore mother-cell has a segregated nucleolus in which the dense fibrillar c o m p o n e n t occupies the center and the granular c o m p o n e n t the periphery (Fig. 1). This segregated m o r p h o l o g y persists throughout prophase I until the nucleolus is fully dispersed (Figs. 1-6). In the preleptotene stage, the fibrillar centers are seen inside the dense fibrillar c o m p o n e n t , s h o w i n g u l t r a s t r u c t u r a l features identical to those we have cytochemically described as "heterogeneous fibrillar centers" in somatic cells (Risuefio et al., 1982). They have small inclusions o f condensed chromatin intermingled with a lighter material which contains decondensed chromatin and proteins (Fig. 1). M o r p h o m e t r i cally, the fibrillar centers are large at this stage and their n u m b e r per nucleolar section is very low, c o m p a r e d to other stages (Fig.
15). In the leptotene stage, the inner dense fibrillar c o m p o n e n t o f the segregated nucleolus also contains fibrillar centers in its interior; however, these show fewer inclusions o f condensed c h r o m a t i n than in the preceding stage, and nearly all the space is occupied only by the lighter fibrous material (Fig. 2). The cross-sectional area is comparatively smaller, and their n u m b e r higher (Fig. 15).
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FIG. 1. Allium cepa pollen mother-cell. Preleptotene stage just before meiosis. Segregated nucleolus showing a large fibrillar center (arrow) inside the dense fibrillar component (F). It has a heterogeneous structure, with dark chromatin inclusions immersed in a lighter fibrous material. G, nucleolar granular component, x 18 700. FIG. 2. Allium cepa pollen mother-cell. Leptotene stage. Segregated nucleolus with several fibrillar centers variable in shape, size, and structure. In some cases, dark chromatin cores are present (arrow), while other fibrillar centers are fully homogeneous (arrowhead). F, nucleolar dense fibrillar component; G, nucleolar granular component, x 18 000.
During middle prophase I (zygotene and pachytene) practically no discrete fibrillar centers can be resolved, but the nucleolar o r g a n i z e r ( N O R ) is a l w a y s s e e n at t h e p e riphery of the nucleolus, associated with masses of extranucleolar condensed ehrom a t i n (Figs. 3 - 5 ) a n d a l s o j o i n e d t o " n u -
cleolar dense bodies" (Medina and Risuefio, 1981) (Fig. 3). T h e r e is a s t r o n g d i f f e r e n c e between the structure of the NOR and that of the condensed extranucleolar chromatin m a s s e s w h i c h a r e a s s o c i a t e d w i t h it: n o c h r o m a t i n i n c l u s i o n s at all a r e p r e s e n t i n t h e N O R a t t h e s e stages, b u t o n l y t h e m e -
FIGS. 3-5. Middle meiotic prophase I. Segregated nucleolus. The NOR (arrows) is always seen on the periphery of the nucleolar mass associated with masses of condensed extranucleolar chromatin (Chr) and joined to "nucleolar dense bodies" (ndb; Fig. 3), which are the products of its activity. The N O R shows a homogeneous structure. When highly magnified (Fig. 5), denser 8- to 10-rim fibers (small arrows) can be differentiated from lighter fibrils. Figure 3: A. cepa pollen mother-cell in pachytene. Figures 4 and 5: Pisurn sativum megaspore mother-cell in zygotene. F, Nucleolar dense fibrillar component; G, nucleolar granular component; SC, synaptonemal complexes. Fig. 3, × 26 300; Fig. 4, x 18 500, and Fig. 5, × 44000. FIG. 6. Allium cepa pollen mother-cell. Diakinesis stage. The nucleolus is at the end of its dispersion process and consists of two round-shaped masses between which is the NOR (arrows). The NOR structure is heterogeneous, with dark chromatin inclusions and lighter fibrous material. Chr, chromosomes. × 30 800.
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dium electron-dense material which, when highly magnified, is seen to be made up o f denser chromatin fibers 8-10 n m in diameter and surrounded by other lighter ones (Fig. 5). This structure is identical to that which we have cytochemically described in somatic cells as " h o m o g e n e o u s fibrillar centers" (Risuefio e t al., 1982). As prophase progresses in the diplotene stage, some inclusions o f condensed c h r o m a t i n appear, giving the N O R a structure "intermediate" between the h o m o g e n e o u s and heterogeneous types. In the diakinesis stage, the nucleolus is finishing its dispersion, and can be seen in sections in the form o f two nucleolar masses, the N O R being between them. It has a heterogeneous structure with relatively large, condensed chromatin inclusions (Fig. 6). In metaphase I, the N O R is recognizable as a special segment o f c h r o m o s o m e s , different in structure from the nonnucleolar chromatin that is heavily electron opaque, and also different from the kinetochores, which are easily identifiable by their position (Fig. 7). The N O R shows a heterogen e o u s s t r u c t u r e closely r e s e m b l i n g the structure observed in preleptotene and diakinesis (Fig. 7). At the end o f the meiosis, the nucleolus reorganizes. In telophase II and the tetrad stage, the nucleolus is usually seen in thin sections as two r o u n d e d masses between which is the N O R (Fig. 8). T h r o u g h o u t the early G1 period o f microspore interphase, it is possible to follow the evolution o f the
structure o f the N O R , which steadily changes from corresponding to the "heterogeneous" type to the " h o m o g e n e o u s " one. This occurs simultaneously with the nucleolar development; the nucleolar masses lose their r o u n d e d shape and gradually b e c o m e m o r e and m o r e irregular (Figs. 8-10). During spore middle interphase, the nucleolus again becomes a single, r o u n d e d c o m p a c t mass. Fibrillar centers are seen within the dense fibrillar c o m p o n e n t , showing a " h o m o g e n e o u s " structure without condensed c h r o m a t i n inclusions in their interior (Fig. 11). During this period, the fibrillar centers are small and their n u m b e r per nucleolar section is relatively high (Fig. 15). At the end o f interphase, the spore nucleolus is vacuolated with fibrillar centers and the fibrillar c o m p o n e n t forming a cortex surrounding a large central vacuole (Fig. 12). A n extremely high n u m b e r o f fibrillar centers per nucleolar section can be seen in this period; their size is the smallest found throughout all the gametogenic stages (Fig. 15). They have a " h o m o g e n e o u s " structure. After the first postmeiotic mitosis, there are two cells in the pollen grain, one generative and the other vegetative. In the generative cell, the nucleolus remains with a mostly fibrillar structure after the end o f mitosis (Fig. 13). The fibrillar centers in this cell are the largest found in the gametogenic process, while the n u m b e r per nucleolar section is the lowest (Fig. 15). They have a "heterogeneous" structure with condensed
FIG. 7. Allium cepa pollen mother-cell. Metaphase 1. The NOR (arrow) is seen as the chromosomal secondary constriction, with a heterogeneousstructure. Note the structural differencesbetween the NOR and the kinetochore (K). Chr, chromosomes, x 16 000. FIGS. 8-10. Allium cepa microspores. Successive steps in the resumption of nucleolar activity after meiosis. The nucleolus at the beginning shows the NOR between two rounded fibrillar masses (F, Fig. 8). The nucleolar masses get progressivelymore irregular in shape (Figs. 9 and 10). At the same time, the NOR (arrows), which at first is strongly heterogeneous (Fig. 8), undergoes the decondensation of the chromatin inclusions (Fig. 9) and reaches a homogeneous structure (Fig. 10). Chr, extranucleolar chromatin. Fig. 8, x 18 500; Figs. 9 and 10, × 20 000. FIo. 11. Pisum sativum megaspore in a phase of the G1 period later than that of the microspore in Fig. 10. The nucleolus is a single, rounded mass with dense fibrillar (F) and granular (G) components. Small homogeneous fibrillar centers are found within the fibrillar component (arrows). The extranucleolar chromatin (Chr) presents a high degree of decondensation, x 21 800.
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THE NOR DURING PLANT GAMETOGENESIS chromatin inclusions (Fig. 13). The fibrillar centers can be seen in sections, either in a peripheral or internal position with respect to the nucleolar mass. The relatively frequent peripheral position o f fibrillar centers in this stage is due to their large size, which enables us to easily observe, in sections, the connection between both the intranucleolar and extranucleolar chromatin. In any case, the presence o f internal fibrillar centers withdraws the possibility o f a fully peripheral N O R in this stage (Fig. 13). In contrast, the nucleolus o f the vegetative cell has a structure typical o f a reorganized active plant nucleolus (Fig. 14), similar to that o f the spore middle interphase. N o t only are the m o r p h o m e t r i c data on fibrillar centers in this cell very close to those obtained for middle inter-phase spores (Fig. 15), but their structure is very similar as well, clearly corresponding to the " h o m o g e n e o u s " type (Fig. 14). DISCUSSION Structure-Function Relationship It is known that there is a high nucleolar R N A synthesis in premeiosis which drops just before leptotene (Das, 1965; Sauter, 1969; Porter et al., 1982). T h r o u g h o u t prophase I, the nucleolus does not show labeling after incorporation o f radioactive precursors (Das, 1965), but in zygotene and pachytene the nucleolar D N A is active in the production o f ribosomal precursors, which are stored as "nucleolar dense bodies" (Williams et al., 1973; Medina and Risuefio, 1981). These bodies could account
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for the nucleolar labeling observed by Porter et al. (1982) after tritiated uridine incorporation. Between premeiosis and zygotene, leptotene is an intermediate stage in r D N A transcriptional activity (Sauter, 1971; Porter et al., 1982). In metaphase I, nucleolar synthesis is fully suspended (Sauter, 1971). T h o u g h physiological studies after meiosis are not very detailed, it can be concluded that nucleolar synthesis is slowly resumed in microspores, increasing progressively throughout interphase until it reaches a high peak in G2, just before microspore mitosis (Bryan, 1951; Taylor, 1953; Steffensen, 1966; Mascarenhas, 1971, 1975). This peak is morphologically expressed by a vacuolated nucleolus, which is related to an increased nucleolar activity, as we have shown elsewhere (Moreno Diaz de la Espina et al., 1980). After first postmeiotic mitosis, the generative cell remains extremely low in activity, in contrast to the vegetative cell whose activity is high (Woodard, 1958; M a s c a r e n h a s a n d Bell, 1970; J a l o u z o t , 1969). By comparing these data on nucleolar activity with the ultrastructure and m o r p h o metric features o f fibrillar centers reported in this paper, we can extend to germinal cells the statements demonstrated by ourselves in somatic plant cells (Risuefio et al., 1982; Medina et al., 1983). There are two main structural types o f fibrillar centers, depending on the degree o f condensation o f the nucleolar chromatin: homogeneous, with only decondensed chromatin; and heterogeneous, with both condensed and decondensed chromatin. These two types are cor-
FIG. 12. Hyacinthoides non-scriptus microspore in the G2 interphase period. Nucleolus with a large central vacuole (V) in which clusters of granules can be found (small arrows). In the nucleolar cortex (Cx) very many small, homogeneous fibrillar centers (arrows) are present. G, granular component; Ex, exine, x 14600. FIc. 13. Hyacinthoides non-scriptus generative cell of a bicellular pollen grain. The nucleolus is exclusively fibriUar in structure (F), and has large, heterogeneous fibrillar centers (arrows). Due to their large size, it is easy to observe in the sections the connection between both the intranucleolar and extranucleolar chromatin (arrowhead). Chr, extranucleolar chromatin, x 21000. FIr. 14. Hyacinthoides non-scriptus vegetative ceU of a bicellular pollen grain. The nucleolus is made up of dense fibrillar (F) and granular (G) components. Small homogeneous fibrillar centers (arrows) are seen inside the fibrillar part. x 13400.
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FIG. 15. Graphical representation of the morphometric features offlbrillar centers throughout gametogenesis. (A) Size of fibrillar centers, measured as the cross-sectionalarea in micrometers squared. (B) Number of fibrillar centers per nucleolar section. In both graphs, columns indicate mean values for each stage, and thin lines, standard deviation values. Abscissa: stage ofgametogenesis(PL, preleptotene; L, leptotene; LG1, late G1 period of spore inter-phase; G2, G2 period of spore interphase; GC, generative cell of bicellular pollen grain; VC, vegetative cell of bicellular pollen grain). Stages between leptotene and late G 1 of the spore are not represented because the concept "fibrillar center" can hardly be used in them.
related with active and low-active nucleoli, respectively. Medium-active nucleoli (as can be the case o f the leptotene and diplotene nucleoli) have fibrillar centers "intermediate" between the two main types. The evolution from the "heterogeneous" to the "hom o g e n e o u s " structure can be clearly seen in the earliest microspore, when the nucleolus is resuming its activity after meiosis. This also demonstrates that both types o f structure are actually different stages o f the same component. Morphometrically, the more active a nucleolus is, the higher the n u m b e r offibrillar centers, and the smaller they are. As we have demonstrated (Medina et aL, 1983), the conclusions obtained after measurements on r a n d o m sections o f nucleoli are a good estimate o f the actual data ob-
tained after tridimensional reconstruction from serial sections. The correlation between the structural changes o f fibrillar centers and the variations o f N O R activity t h r o u g h o u t gametogenesis, according to the available data, is shown in Fig. 16.
Fibrillar Centers a n d N O R In metaphase I, the transcriptional activity o f the N O R is fully suspended; so, at this stage, the classically referred to " s e c o n d a r y constriction" o f the c h r o m o s o m e s contains the whole set o f ribosomal cistrons in an inactive state. Thus, the structure o f these "secondary constrictions" should be a good reference for knowing the structure o f large inactive portions o f the N O R . In this way,
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® Stage
LEPTOTENE
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BICELLULAR POLLEN GRAIN -> < GENERATiVECELL VEGETATIVE CELL VERY LOW
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Light fibrous material (including decondensed chromatin ) Condensed chromatin inc]usions
FIG. 16. Schematicdrawing of the evolution ofthe NOR structure throughout gametogenesis,from premeiosis to the bicellular pollen grain, in relationship to the NOR activity (obtained from the available data). It is evident that the stages with low or no activity are characterized by a heterogeneous structure of the fibrillar centers or the whole nucleolar chromatin; when the NOR is active, fibrillar centers are homogeneous. Medium-active stages show intermediate fibrillar centers. Compare with graphs in Fig. 15 to see the clear correlation between both the ultrastructure and morphometric features of fibrillar centers and the nucleolar activity.
the identity o f structure between the N O R in metaphase I, and fibrillar centers during low-activity stages (such as preleptotene, diakinesis, early G1 spore, and generative cell), supports the view o f fibrillar centers as structural counterparts o f those portions o f the N O R being temporarily inactive (Goessens and Lepoint, 1979; Fakan and Puvion, 1980; Mirre and Stahl, 1981; Risuefio et al., 1982).
The Special Case of Zygotene-Pachytene The location o f fibrillar centers in the nucleolus does not depend on nucleolar activity. However, there is a special case in which the N O R , as a whole, is always seen at the periphery o f the nucleolar mass: namely, middle meiotic prophase I (zygotene and pachytene, Figs. 3 and 4). Other authors have explained this fact as being the result o f the shift o f the N O R from the interior to the surface o f the nucleolus (due to chromatin condensation, which causes its retraction) and as being related to N O R inactivation (Esponda and Gim6nez-Martin, 1975; G i m 6 n e z - M a r t i n et al., 1977; J o r d a n
and Luck, 1976). In view o f our results the shift does occur; but these reasons should not be used for explaining this special case. I f the intranucleolar c h r o m a t i n were strongly condensed, it would not be possible to find a " h o m o g e n e o u s " structure with only 8- to 10-nm c h r o m a t i n fibers, as is the case in zygotene and pachytene. On the other hand, the N O R is active in middle prophase I, and its activity is directed toward the production o f "nucleolar dense bodies" (Williams et al., 1973; Medina and Risuefio, 1981; Porter et al., 1982). Therefore, the fine structure o f the N O R in pachytene follows the pattern o f the relationships between ultrastructure and activity exactly as reported above. In conclusion, the structure-function relationship in plant cells is clearly evident in the nucleolar organizer, since every stage o f nucleolar activity has its own expression in the structure o f fibrillar centers as well as in their m o r p h o m e t r i c features.
Note. The nucleolus nomenclature in this paper follows the recommendation agreed on at the Eighth Nucleolar Workshop (Banyuls-sur-Mer, 1983). With ref-
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erence to our previous papers on the subject, we now use "dense fibrillar component" instead of "fibrillar component," and "condensed chromatin inclusions" (in heterogeneous fibrillar centers) instead of "condensed chromatin cores." We thank Mr. S. Jones for checking the English style of the text. This work has been supported by the CAICYT Grant 3753/79 C2 and by the CSIC Grant 41123. REFERENCES BERTOU% M. (1983) Biol. Cell. 48, 12a. BRYAN, J. H. D. (1951) Chromosoma 4, 369-392. DAs, N. K. (1965) Exp. CellRes. 40, 360-364. ESPONDA, P., AND GIMI~NEZ-MARTiN, G. (1974) Chromosoma 45, 203-213. ESPONDA, P., AND GIMI~NEZ-MART~N,G. (1975) Chromosoma 52, 73-87. FAKAN, S., AND PUVlON, E. (1980) Int. Rev. Cytol. 65, 255-299. GIMt~NEZ-MARTiN, G., DE LA TORRE, C., LOPEZ-SAEZ, J. F., AND ESPONDA, P. (1977) Cytobiologie 14, 421462. GOESSENS, G., AND LEPOINT, A. (1979) Biol. Cell. 35, 211-220. INGLE, J., AND SINCLAIR, J. (1972) Nature (London) 235, 30-32. JALOUZOT, R. (1969) Exp. Cell Res. 55, 1-8. JORDAN, E. G., AND LUCK, B. T. (1976) J. CellSci. 22, 75-86.
MASCARENHAS, J. P. (1971) in HESLOP-HARRISON, J. (Ed.), Pollen: Development and Physiology, pp. 201222, Butterworths, London. MASCARENHAS, J. P. (1975) Bot. Rev. 41, 259-313. MASCARENHAS, J. P., AND BELL, E. (1970) Dev. Biol. 21, 475-490. MEDINA, F. J., AND RISUENO, M. C. (1981) BioL Cell. 42, 79-86. MEDINA, F. J., RISUENO, M. C., AND MORENO D~AZDE LA ESPINA, S. (1983) Biol. Cell. 48, 31-38. MIRRE, C., AND STAHL, A. (1981) J. CellSci. 48, 105126. MORENO DiAZ DE LA ESPINA, S. (1976) Experientia 32, 1384-1386. MORENO Diaz DE LA ESPINA, S., MEDINA, F. J., AND RISUENO, M. C. (1980) Eur. J. Cell Biol. 22, 724729. PORTER, E. K., BIRD, J. M., AND DICKINSON, H. G. (1982) J. Cell Sci. 57, 229-246. RISUEf40, M. C., MEDINA, F. J., AND MORENO DiAZ DE rA ESPINA, S. (1982) J. Cell Sci. 58, 313-329. SAUTER, J. J. (1969) Z. Pflanzenphysiol. 61, 1-19. SAUTER, J. J. (1971) in HESLOP-HARRISON, J. (Ed.), Pollen: Development and Physiology, pp. 3-15, Butterworths, London. STEFFENSEN, D. M. (1966) Exp. Cell Res. 44, 1-12. TAYLOR, J. H. (1953) Exp. CellRes. 4, 164-173. WILLIAMS, E., HESLOP-HARRISON, J., AND DICKINSON, H. G. (1973) Protoplasma 77, 79-93. WOODARD, J. W. (1958) J. Biophys. Biochem. Cytol. 4, 383-390.
(1983)
The Nucleolar Organizer (NOR) and Fibrillar Centers during Plant Gametogenesis F. J. MEDINA, 1 M. C. RISUENO, M. I. RODRIGUEZ-GARCIA, 2 AND M. A. SANCHEz-PINA Morfogdnesis Celular, Instituto de Biologfa Celular, CSIC, Velgtzquez 144, Madrid-6, Spain Received April 27, 1983 The ultrastructure and morphometric features of nucleolar fibrillar centers have been studied in three different plant species over a long period of gametogenesis, extending from premeiosis to after the first postmeiotic mitosis, with respect to variations in nucleolar activity which occur throughout this process. Fibrillar centers represent the structural counterpart of the transcriptionally inactive portions of the nucleolar organizing region (NOR); in some gametogenic stages, no discrete fibrillar centers can be observed and, therefore, the structure of the whole NOR has been studied. Two clearly differentiated types of fibrillar center structure are described, with a close relationship to different stages of nucleolar activity. Stages with high activity show "homogeneous" fibrillar centers, whose cbromatin is decondensed, and stages with low activity show "heterogeneous" fibrillar centers with both condensed and decondensed chromatin. These two types had previously been characterized cytochemically by us in somatic cells. Medium-active stages show fibrillar centers "intermediate" between the two main types. The morphometric behavior of fibrillar centers is also related to activity. The number of fibrillar centers per nucleolar section increases with the nucleolar activity, while their size decreases. However, the location of fibrillar centers in the nucleolus does not follow any special pattern, except in middle meiotic prophase I, in which the whole NOR occupies a peripheral position. This is due to a special NOR activity at these stages, whose products are stored as "nueleolar dense bodies." All these data allow us to conclude that fibrillar centers are highly dynamic structures, extremely sensitive to changes in nucleolar activity.
In a previous paper (Risuefio et al., 1982) we have shown that, in plant meristematic cells, the nucleolar fibrillar centers are made up of DNA and proteins, this DNA being inactive in transcription. The transcriptionally active nucleolar DNA is found in the dense fibrillar component. Both fibrillar centers and dense fibrillar component constitute a functional unit (Moreno Diaz de la Espina, 1976), being the structural counterpart of the nucleolar organizing region (NOR). So, the plant cell fibrillar centers are functionally identical to the animal cell ones (Goessens and Lepoint, 1979; Fakan and Puvion, 1980; Mirre and Stahl, 1981). However, the fibrillar centers of plant meristematic cells undergo changes that depend on the nucleolar activity. These changes afTo whom correspondence should be addressed. 2 Present address: Estaci6n Experimental del Zaidin (CSIC), Profesor Albareda, 1, Granada, Spain.
fect the ultrastructure and the morphometric features (number and size) of fibrillar centers. From a morphometric point of view, fibrillar centers are small and numerous in highly active nucleoli and larger and fewer as nucleolar activity drops (Medina et al., 1983). Ultrastructurally, in experimental conditions of reduced or suspended nucleolar activity, inclusions of condensed chromatin are seen in fibrillar centers, which are not present when nucleolar activity is high. This fact defines two types of fibrillar centers in plant cells, correlated with nucleolar activity: homogeneous, resembling very much those described in animal cells, and heterogeneous, which, in addition, contain condensed chromatin inclusions (Risuefio et al., 1982). This latter type is not usual in animal cells; it has only been reported in Nereis ovocytes (Bertout, 1983). Its existence in plants is most probably due to the number of ribosomal cistrons, much higher 300
0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
THE NOR DURING PLANT GAMETOGENESIS in plant than in animal cells (Ingle and Sinclair, 1972). In order to demonstrate whether or not this behavior o f fibrillar centers is general in plant cells we have l o o k e d for a plant cellular system that is completely different from the root meristem, and which has strong variations in its nucleolar activity under natural conditions. The system we have chosen is that o f reproducing tissues t h r o u g h o u t gametogenesis. During this process, the germinal cells pass, in a short time, through successive periods o f increased and decreased activity. The structure and activity o f the nucleolar organizer have been studied in a few stages o f plant male gametogenesis (Williams et aL, 1973; Esponda and G i m r n e z - M a r t l n , 1974, 1975; J o r d a n and Luck, 1976; Gim6nez-Martin et aL, 1977), but it has not been possible to reach any general conclusions on the structure-function relationship from these studies. For this reason, we have chosen a long period o f plant gametogenesis, extending from premeiosis up to the stages that c o m e after the first postmeiotic mitosis in both the male and female reproducing cells. During this period, the fibrillar centers undergo strong morphologic changes which we have related to the very different levels o f nucleolar activity that exist from one stage to another. Furthermore, the use o f three different species enables us to obtain conclusions o f b r o a d application for plant cells. MATERIALS AND METHODS Ovaries from Pisum sativum L. and anthers from Allium cepa and Hyacinthoides non-scriptus, all belonging to flowers grown in a greenhouse, were dissected from flower buds. For checking the stage of development, one anther from each flower was squashed in 2% acetic orcein. In the case of the ovaries, checking was only possible on semithin sections of fixed and embedded samples. Anthers were fixed with 3% glutaraldehyde in 0.05 Mcacodylate buffer for 2 hr at room temperature. The fixation of ovaries was carried out in the same way, except that the molarity of the buffer was 0.025 M cacodylate. Both anthers and ovaries were postfixed with 1%
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OsO4, in the same buffer that was used for glutaraldehyde fixation, for 1 hr at room temperature, then dehydrated in ethanol and embedded in Epon 812. Semithin sections were examined under the light microscope prior to ultramicrotomy. Ultrathin sections were cut on an LKB Ultrotome III, stained with uranyl acetate and lead citrate, and then observed under a Philips 300 electron microscope. Morphometry of Random Sections
We have calculated the number of fibrillar centers per section and their cross-sectional areas in those gametogenic stages displaying discrete fibrillar centers. Measurements of areas were carried out with a Kentron MOP/AM03 semiautomatic image analyzer. For both features in every stage, mean and standard deviation values were calculated and graphically represented. RESULTS Just before the beginning o f meiosis, the newly differentiated spore mother-cell has a segregated nucleolus in which the dense fibrillar c o m p o n e n t occupies the center and the granular c o m p o n e n t the periphery (Fig. 1). This segregated m o r p h o l o g y persists throughout prophase I until the nucleolus is fully dispersed (Figs. 1-6). In the preleptotene stage, the fibrillar centers are seen inside the dense fibrillar c o m p o n e n t , s h o w i n g u l t r a s t r u c t u r a l features identical to those we have cytochemically described as "heterogeneous fibrillar centers" in somatic cells (Risuefio et al., 1982). They have small inclusions o f condensed chromatin intermingled with a lighter material which contains decondensed chromatin and proteins (Fig. 1). M o r p h o m e t r i cally, the fibrillar centers are large at this stage and their n u m b e r per nucleolar section is very low, c o m p a r e d to other stages (Fig.
15). In the leptotene stage, the inner dense fibrillar c o m p o n e n t o f the segregated nucleolus also contains fibrillar centers in its interior; however, these show fewer inclusions o f condensed c h r o m a t i n than in the preceding stage, and nearly all the space is occupied only by the lighter fibrous material (Fig. 2). The cross-sectional area is comparatively smaller, and their n u m b e r higher (Fig. 15).
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FIG. 1. Allium cepa pollen mother-cell. Preleptotene stage just before meiosis. Segregated nucleolus showing a large fibrillar center (arrow) inside the dense fibrillar component (F). It has a heterogeneous structure, with dark chromatin inclusions immersed in a lighter fibrous material. G, nucleolar granular component, x 18 700. FIG. 2. Allium cepa pollen mother-cell. Leptotene stage. Segregated nucleolus with several fibrillar centers variable in shape, size, and structure. In some cases, dark chromatin cores are present (arrow), while other fibrillar centers are fully homogeneous (arrowhead). F, nucleolar dense fibrillar component; G, nucleolar granular component, x 18 000.
During middle prophase I (zygotene and pachytene) practically no discrete fibrillar centers can be resolved, but the nucleolar o r g a n i z e r ( N O R ) is a l w a y s s e e n at t h e p e riphery of the nucleolus, associated with masses of extranucleolar condensed ehrom a t i n (Figs. 3 - 5 ) a n d a l s o j o i n e d t o " n u -
cleolar dense bodies" (Medina and Risuefio, 1981) (Fig. 3). T h e r e is a s t r o n g d i f f e r e n c e between the structure of the NOR and that of the condensed extranucleolar chromatin m a s s e s w h i c h a r e a s s o c i a t e d w i t h it: n o c h r o m a t i n i n c l u s i o n s at all a r e p r e s e n t i n t h e N O R a t t h e s e stages, b u t o n l y t h e m e -
FIGS. 3-5. Middle meiotic prophase I. Segregated nucleolus. The NOR (arrows) is always seen on the periphery of the nucleolar mass associated with masses of condensed extranucleolar chromatin (Chr) and joined to "nucleolar dense bodies" (ndb; Fig. 3), which are the products of its activity. The N O R shows a homogeneous structure. When highly magnified (Fig. 5), denser 8- to 10-rim fibers (small arrows) can be differentiated from lighter fibrils. Figure 3: A. cepa pollen mother-cell in pachytene. Figures 4 and 5: Pisurn sativum megaspore mother-cell in zygotene. F, Nucleolar dense fibrillar component; G, nucleolar granular component; SC, synaptonemal complexes. Fig. 3, × 26 300; Fig. 4, x 18 500, and Fig. 5, × 44000. FIG. 6. Allium cepa pollen mother-cell. Diakinesis stage. The nucleolus is at the end of its dispersion process and consists of two round-shaped masses between which is the NOR (arrows). The NOR structure is heterogeneous, with dark chromatin inclusions and lighter fibrous material. Chr, chromosomes. × 30 800.
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dium electron-dense material which, when highly magnified, is seen to be made up o f denser chromatin fibers 8-10 n m in diameter and surrounded by other lighter ones (Fig. 5). This structure is identical to that which we have cytochemically described in somatic cells as " h o m o g e n e o u s fibrillar centers" (Risuefio e t al., 1982). As prophase progresses in the diplotene stage, some inclusions o f condensed c h r o m a t i n appear, giving the N O R a structure "intermediate" between the h o m o g e n e o u s and heterogeneous types. In the diakinesis stage, the nucleolus is finishing its dispersion, and can be seen in sections in the form o f two nucleolar masses, the N O R being between them. It has a heterogeneous structure with relatively large, condensed chromatin inclusions (Fig. 6). In metaphase I, the N O R is recognizable as a special segment o f c h r o m o s o m e s , different in structure from the nonnucleolar chromatin that is heavily electron opaque, and also different from the kinetochores, which are easily identifiable by their position (Fig. 7). The N O R shows a heterogen e o u s s t r u c t u r e closely r e s e m b l i n g the structure observed in preleptotene and diakinesis (Fig. 7). At the end o f the meiosis, the nucleolus reorganizes. In telophase II and the tetrad stage, the nucleolus is usually seen in thin sections as two r o u n d e d masses between which is the N O R (Fig. 8). T h r o u g h o u t the early G1 period o f microspore interphase, it is possible to follow the evolution o f the
structure o f the N O R , which steadily changes from corresponding to the "heterogeneous" type to the " h o m o g e n e o u s " one. This occurs simultaneously with the nucleolar development; the nucleolar masses lose their r o u n d e d shape and gradually b e c o m e m o r e and m o r e irregular (Figs. 8-10). During spore middle interphase, the nucleolus again becomes a single, r o u n d e d c o m p a c t mass. Fibrillar centers are seen within the dense fibrillar c o m p o n e n t , showing a " h o m o g e n e o u s " structure without condensed c h r o m a t i n inclusions in their interior (Fig. 11). During this period, the fibrillar centers are small and their n u m b e r per nucleolar section is relatively high (Fig. 15). At the end o f interphase, the spore nucleolus is vacuolated with fibrillar centers and the fibrillar c o m p o n e n t forming a cortex surrounding a large central vacuole (Fig. 12). A n extremely high n u m b e r o f fibrillar centers per nucleolar section can be seen in this period; their size is the smallest found throughout all the gametogenic stages (Fig. 15). They have a " h o m o g e n e o u s " structure. After the first postmeiotic mitosis, there are two cells in the pollen grain, one generative and the other vegetative. In the generative cell, the nucleolus remains with a mostly fibrillar structure after the end o f mitosis (Fig. 13). The fibrillar centers in this cell are the largest found in the gametogenic process, while the n u m b e r per nucleolar section is the lowest (Fig. 15). They have a "heterogeneous" structure with condensed
FIG. 7. Allium cepa pollen mother-cell. Metaphase 1. The NOR (arrow) is seen as the chromosomal secondary constriction, with a heterogeneousstructure. Note the structural differencesbetween the NOR and the kinetochore (K). Chr, chromosomes, x 16 000. FIGS. 8-10. Allium cepa microspores. Successive steps in the resumption of nucleolar activity after meiosis. The nucleolus at the beginning shows the NOR between two rounded fibrillar masses (F, Fig. 8). The nucleolar masses get progressivelymore irregular in shape (Figs. 9 and 10). At the same time, the NOR (arrows), which at first is strongly heterogeneous (Fig. 8), undergoes the decondensation of the chromatin inclusions (Fig. 9) and reaches a homogeneous structure (Fig. 10). Chr, extranucleolar chromatin. Fig. 8, x 18 500; Figs. 9 and 10, × 20 000. FIo. 11. Pisum sativum megaspore in a phase of the G1 period later than that of the microspore in Fig. 10. The nucleolus is a single, rounded mass with dense fibrillar (F) and granular (G) components. Small homogeneous fibrillar centers are found within the fibrillar component (arrows). The extranucleolar chromatin (Chr) presents a high degree of decondensation, x 21 800.
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THE NOR DURING PLANT GAMETOGENESIS chromatin inclusions (Fig. 13). The fibrillar centers can be seen in sections, either in a peripheral or internal position with respect to the nucleolar mass. The relatively frequent peripheral position o f fibrillar centers in this stage is due to their large size, which enables us to easily observe, in sections, the connection between both the intranucleolar and extranucleolar chromatin. In any case, the presence o f internal fibrillar centers withdraws the possibility o f a fully peripheral N O R in this stage (Fig. 13). In contrast, the nucleolus o f the vegetative cell has a structure typical o f a reorganized active plant nucleolus (Fig. 14), similar to that o f the spore middle interphase. N o t only are the m o r p h o m e t r i c data on fibrillar centers in this cell very close to those obtained for middle inter-phase spores (Fig. 15), but their structure is very similar as well, clearly corresponding to the " h o m o g e n e o u s " type (Fig. 14). DISCUSSION Structure-Function Relationship It is known that there is a high nucleolar R N A synthesis in premeiosis which drops just before leptotene (Das, 1965; Sauter, 1969; Porter et al., 1982). T h r o u g h o u t prophase I, the nucleolus does not show labeling after incorporation o f radioactive precursors (Das, 1965), but in zygotene and pachytene the nucleolar D N A is active in the production o f ribosomal precursors, which are stored as "nucleolar dense bodies" (Williams et al., 1973; Medina and Risuefio, 1981). These bodies could account
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for the nucleolar labeling observed by Porter et al. (1982) after tritiated uridine incorporation. Between premeiosis and zygotene, leptotene is an intermediate stage in r D N A transcriptional activity (Sauter, 1971; Porter et al., 1982). In metaphase I, nucleolar synthesis is fully suspended (Sauter, 1971). T h o u g h physiological studies after meiosis are not very detailed, it can be concluded that nucleolar synthesis is slowly resumed in microspores, increasing progressively throughout interphase until it reaches a high peak in G2, just before microspore mitosis (Bryan, 1951; Taylor, 1953; Steffensen, 1966; Mascarenhas, 1971, 1975). This peak is morphologically expressed by a vacuolated nucleolus, which is related to an increased nucleolar activity, as we have shown elsewhere (Moreno Diaz de la Espina et al., 1980). After first postmeiotic mitosis, the generative cell remains extremely low in activity, in contrast to the vegetative cell whose activity is high (Woodard, 1958; M a s c a r e n h a s a n d Bell, 1970; J a l o u z o t , 1969). By comparing these data on nucleolar activity with the ultrastructure and m o r p h o metric features o f fibrillar centers reported in this paper, we can extend to germinal cells the statements demonstrated by ourselves in somatic plant cells (Risuefio et al., 1982; Medina et al., 1983). There are two main structural types o f fibrillar centers, depending on the degree o f condensation o f the nucleolar chromatin: homogeneous, with only decondensed chromatin; and heterogeneous, with both condensed and decondensed chromatin. These two types are cor-
FIG. 12. Hyacinthoides non-scriptus microspore in the G2 interphase period. Nucleolus with a large central vacuole (V) in which clusters of granules can be found (small arrows). In the nucleolar cortex (Cx) very many small, homogeneous fibrillar centers (arrows) are present. G, granular component; Ex, exine, x 14600. FIc. 13. Hyacinthoides non-scriptus generative cell of a bicellular pollen grain. The nucleolus is exclusively fibriUar in structure (F), and has large, heterogeneous fibrillar centers (arrows). Due to their large size, it is easy to observe in the sections the connection between both the intranucleolar and extranucleolar chromatin (arrowhead). Chr, extranucleolar chromatin, x 21000. FIr. 14. Hyacinthoides non-scriptus vegetative ceU of a bicellular pollen grain. The nucleolus is made up of dense fibrillar (F) and granular (G) components. Small homogeneous fibrillar centers (arrows) are seen inside the fibrillar part. x 13400.
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FIG. 15. Graphical representation of the morphometric features offlbrillar centers throughout gametogenesis. (A) Size of fibrillar centers, measured as the cross-sectionalarea in micrometers squared. (B) Number of fibrillar centers per nucleolar section. In both graphs, columns indicate mean values for each stage, and thin lines, standard deviation values. Abscissa: stage ofgametogenesis(PL, preleptotene; L, leptotene; LG1, late G1 period of spore inter-phase; G2, G2 period of spore interphase; GC, generative cell of bicellular pollen grain; VC, vegetative cell of bicellular pollen grain). Stages between leptotene and late G 1 of the spore are not represented because the concept "fibrillar center" can hardly be used in them.
related with active and low-active nucleoli, respectively. Medium-active nucleoli (as can be the case o f the leptotene and diplotene nucleoli) have fibrillar centers "intermediate" between the two main types. The evolution from the "heterogeneous" to the "hom o g e n e o u s " structure can be clearly seen in the earliest microspore, when the nucleolus is resuming its activity after meiosis. This also demonstrates that both types o f structure are actually different stages o f the same component. Morphometrically, the more active a nucleolus is, the higher the n u m b e r offibrillar centers, and the smaller they are. As we have demonstrated (Medina et aL, 1983), the conclusions obtained after measurements on r a n d o m sections o f nucleoli are a good estimate o f the actual data ob-
tained after tridimensional reconstruction from serial sections. The correlation between the structural changes o f fibrillar centers and the variations o f N O R activity t h r o u g h o u t gametogenesis, according to the available data, is shown in Fig. 16.
Fibrillar Centers a n d N O R In metaphase I, the transcriptional activity o f the N O R is fully suspended; so, at this stage, the classically referred to " s e c o n d a r y constriction" o f the c h r o m o s o m e s contains the whole set o f ribosomal cistrons in an inactive state. Thus, the structure o f these "secondary constrictions" should be a good reference for knowing the structure o f large inactive portions o f the N O R . In this way,
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FIG. 16. Schematicdrawing of the evolution ofthe NOR structure throughout gametogenesis,from premeiosis to the bicellular pollen grain, in relationship to the NOR activity (obtained from the available data). It is evident that the stages with low or no activity are characterized by a heterogeneous structure of the fibrillar centers or the whole nucleolar chromatin; when the NOR is active, fibrillar centers are homogeneous. Medium-active stages show intermediate fibrillar centers. Compare with graphs in Fig. 15 to see the clear correlation between both the ultrastructure and morphometric features of fibrillar centers and the nucleolar activity.
the identity o f structure between the N O R in metaphase I, and fibrillar centers during low-activity stages (such as preleptotene, diakinesis, early G1 spore, and generative cell), supports the view o f fibrillar centers as structural counterparts o f those portions o f the N O R being temporarily inactive (Goessens and Lepoint, 1979; Fakan and Puvion, 1980; Mirre and Stahl, 1981; Risuefio et al., 1982).
The Special Case of Zygotene-Pachytene The location o f fibrillar centers in the nucleolus does not depend on nucleolar activity. However, there is a special case in which the N O R , as a whole, is always seen at the periphery o f the nucleolar mass: namely, middle meiotic prophase I (zygotene and pachytene, Figs. 3 and 4). Other authors have explained this fact as being the result o f the shift o f the N O R from the interior to the surface o f the nucleolus (due to chromatin condensation, which causes its retraction) and as being related to N O R inactivation (Esponda and Gim6nez-Martin, 1975; G i m 6 n e z - M a r t i n et al., 1977; J o r d a n
and Luck, 1976). In view o f our results the shift does occur; but these reasons should not be used for explaining this special case. I f the intranucleolar c h r o m a t i n were strongly condensed, it would not be possible to find a " h o m o g e n e o u s " structure with only 8- to 10-nm c h r o m a t i n fibers, as is the case in zygotene and pachytene. On the other hand, the N O R is active in middle prophase I, and its activity is directed toward the production o f "nucleolar dense bodies" (Williams et al., 1973; Medina and Risuefio, 1981; Porter et al., 1982). Therefore, the fine structure o f the N O R in pachytene follows the pattern o f the relationships between ultrastructure and activity exactly as reported above. In conclusion, the structure-function relationship in plant cells is clearly evident in the nucleolar organizer, since every stage o f nucleolar activity has its own expression in the structure o f fibrillar centers as well as in their m o r p h o m e t r i c features.
Note. The nucleolus nomenclature in this paper follows the recommendation agreed on at the Eighth Nucleolar Workshop (Banyuls-sur-Mer, 1983). With ref-
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erence to our previous papers on the subject, we now use "dense fibrillar component" instead of "fibrillar component," and "condensed chromatin inclusions" (in heterogeneous fibrillar centers) instead of "condensed chromatin cores." We thank Mr. S. Jones for checking the English style of the text. This work has been supported by the CAICYT Grant 3753/79 C2 and by the CSIC Grant 41123. REFERENCES BERTOU% M. (1983) Biol. Cell. 48, 12a. BRYAN, J. H. D. (1951) Chromosoma 4, 369-392. DAs, N. K. (1965) Exp. CellRes. 40, 360-364. ESPONDA, P., AND GIMI~NEZ-MARTiN, G. (1974) Chromosoma 45, 203-213. ESPONDA, P., AND GIMI~NEZ-MART~N,G. (1975) Chromosoma 52, 73-87. FAKAN, S., AND PUVlON, E. (1980) Int. Rev. Cytol. 65, 255-299. GIMt~NEZ-MARTiN, G., DE LA TORRE, C., LOPEZ-SAEZ, J. F., AND ESPONDA, P. (1977) Cytobiologie 14, 421462. GOESSENS, G., AND LEPOINT, A. (1979) Biol. Cell. 35, 211-220. INGLE, J., AND SINCLAIR, J. (1972) Nature (London) 235, 30-32. JALOUZOT, R. (1969) Exp. Cell Res. 55, 1-8. JORDAN, E. G., AND LUCK, B. T. (1976) J. CellSci. 22, 75-86.
MASCARENHAS, J. P. (1971) in HESLOP-HARRISON, J. (Ed.), Pollen: Development and Physiology, pp. 201222, Butterworths, London. MASCARENHAS, J. P. (1975) Bot. Rev. 41, 259-313. MASCARENHAS, J. P., AND BELL, E. (1970) Dev. Biol. 21, 475-490. MEDINA, F. J., AND RISUENO, M. C. (1981) BioL Cell. 42, 79-86. MEDINA, F. J., RISUENO, M. C., AND MORENO D~AZDE LA ESPINA, S. (1983) Biol. Cell. 48, 31-38. MIRRE, C., AND STAHL, A. (1981) J. CellSci. 48, 105126. MORENO DiAZ DE LA ESPINA, S. (1976) Experientia 32, 1384-1386. MORENO Diaz DE LA ESPINA, S., MEDINA, F. J., AND RISUENO, M. C. (1980) Eur. J. Cell Biol. 22, 724729. PORTER, E. K., BIRD, J. M., AND DICKINSON, H. G. (1982) J. Cell Sci. 57, 229-246. RISUEf40, M. C., MEDINA, F. J., AND MORENO DiAZ DE rA ESPINA, S. (1982) J. Cell Sci. 58, 313-329. SAUTER, J. J. (1969) Z. Pflanzenphysiol. 61, 1-19. SAUTER, J. J. (1971) in HESLOP-HARRISON, J. (Ed.), Pollen: Development and Physiology, pp. 3-15, Butterworths, London. STEFFENSEN, D. M. (1966) Exp. Cell Res. 44, 1-12. TAYLOR, J. H. (1953) Exp. CellRes. 4, 164-173. WILLIAMS, E., HESLOP-HARRISON, J., AND DICKINSON, H. G. (1973) Protoplasma 77, 79-93. WOODARD, J. W. (1958) J. Biophys. Biochem. Cytol. 4, 383-390.