Planta 133, 15-19 (1976)
9 by Springer-Verlag 1976
Early Embryogenesis in Tropaeolum majus L.: Diversification of Plastids W. Nagl and S. Ktihner Department of Biology, The University of Kaiserslautern, P.O. Box 3049, D-6750 Kaiserslautern, Federal Republic of Germany
Abstract. Embryogeny in the nasturtium is characterized by the development of a large, tripartite suspensor and storing cotyledons. A light and electron microscopic study revealed an early diversification of the plastids in the various regions of the suspensor and the embryo proper. Amyloplasts are found in the developing cotyledons of the heart-like embryo, while chloroplasts occur within the meristematic part of the embryo and the adjacent portion of the suspensor. The cells between the meristem and the storing cotyledons display undifferentiated leukoplasts, whereas leukoplasts with an electron-dense matrix occur in the basal cell mass of the embryo-suspensor. Etioplasts develop in several cells of the placental haustorium of the suspensor. The carpel haustorium shows rather undifferentiated leukoplasts, which are transformed into electron-dense plastids during autolysis of the suspensor. This early plastidal differentiation in discussed with respect to its control and functional significance. Key words: Tropaeolum, Embryogenesis - Differentiation - Plastids - Ultrastructure.
Introduction Early embryogenesis in the nasturtium, Tropaeolum majus L., is characterized by the development of a suspensor, which is m a n y times larger than the embryo proper (Walker, 1947; Nagl, 1962 and in press). The suspensor of Tropaeolum differs from that in other species with the suspensor-cotyledo way of development (Nagl, 1976a) in as far as it is a highly differentiated organ that develops two haustoria into the carpel tissue. Therefore, the study of embryogeny in Tropaeolum may be well suited to elucidate early differentiation processes.
This paper deals with the differentiation of the plastids during early development of the suspensor and the embryo proper. The results indicate different metabolic regulation and function of various parts of this system.
Material and Methods Unripe fruits of Tropaeolum majus L. were harvested, and the embryos together with the suspensors isolated under a dissecting microscope. The material was fixed in 6.25% glutaraldehyde (pH 7.3) for 60 rain, and postfixed in 1% osmium tetroxide for 90 min. After dehydration and block-staining with uranyl acetate, the material was embedded according to Spurr (1969), and studied with a Zeiss EM I0A electron microscope. Semithin sections of the same material which had been used for electron microscopic examination, as well as living ceils, and embryos, which had been fixed in acetic ethanol, were used for light microscopic analysis.
Results The following regions of the suspensor and embryo proper have been studied in detail (Fig. 1): the cotyledons, the meristem of the plumula and radicula, the embryo-suspensor, the basal cell mass, the placental haustorium, and the more distal cells of the chalazal or carpel haustorium. The cotyledons of the heart-like embryo display numerous starch grains (Fig. 2), which can be localized within amyloplasts at the ultrastructural level (Fig. 3). In mature embryos, the cells of the cotyledons are densely filled with starch. The cells between those of the starch-storing cotyldons and the mitotically active cells of the plumula show rather undifferentiated leukoplasts, which often contain a central inclusion or vacuole. The meristematic cells of the plumula and radicula, however, possess distinct chloroplasts with small grana (Fig. 4).
W. Nag1 and S. Ktihner: Diversificationof Plastids
/ Basal Ceil Mass
The plastidal organization in the suspensor is quite different. The cells of the placental haustorium either show undifferentiated leukoplasts, or, particularly in a late heart-like stage of the embryo, typical etioplasts with distinct prolamellar bodies (Fig. 5). The basal cells of the embryo-suspensor, which are located at the exit of the micropyle, exhibit characteristic leukoplasts with an electron-dense matrix and electrontransparent tubes in various directions (Figs. 6, 7). This type of leukoplast is also visible in other suspensor cells, which accumulate lipid bodies, and it becomes very frequent as soon as the suspensor undergoes autolysis (Nagl, unpublished). The elongated cells of the embryo-suspensor as well as the suspensor cells adjacent to the embryo proper display, on the other hand, chloroplasts that are very similar to those in the embryo proper (Figs. 8, 9). They are often found in clusters, from which they seem to be released after growing and dividing (Fig. 8). Typical grana are rarely formed. Both the embryo and the embryo-suspending thread of the suspensor are green in the living state.
In this paper it is shown that plastidal differentiation already occurs during early embryogenesis. It is worthwile discussing this phenomenon from two aspects, the control of this differentiation, and its functional significance. It is well known that the development of plastidal
Fig. 1. Semidiagrammatic drawing of an isolated suspensor and embryo of Tropaeolum majus. The embryo-suspensor elongates through the micropyle and forms a basal cell mass, from which two haustoria originate. The smaller placental haustorium penetrates the carpel tissue at the placenta, the larger carpel haustorium develops within the slip between ovule and carpel and penetrates the maternal tissue at the chalazal pole. The bar indicates 1 mm
ultrastructure and function is controlled by light and phytohormones. Both factors may be involved in the diversification process of the plastids in the embryo and suspensor of Tropaeolum. The light conditions are apparently different for the individual regions, due to the different location of the cells within the fruit. The placental haustorium, on the one hand, penetrates the carpel at its central region, where the carpels are united, and where no light may reach the suspensor cells. Thus, the formation of etioplasts is stimulated. On the other hand, the meristem of the embryo proper, and the cells of the embryo-suspensor are located within the endosperm cavity and micropyle, which are surrounded by rather thin layers of ovule and carpel tissue. Therefore, these portions of the embryo and suspensor are exposed to light and are able to develop chloroplasts. The fact that other parts of the embryo and suspensor, which are exposed to the same light conditions as the meristem and the embryo-suspending thread, develop different plastids, may be explained by some hormonal influences. The effect of, e.g., cytokinin on morphological transformations of plastids has been repeatedly shown (Stetler and Laetsch, 1965; Seyer et al., 1975). Different hormonal levels in the suspensor and embryo have been recently demonstrated in Phaseolus, where hormonal changes during the development have also been established (Alpi et al., 1975; Cionini et al., 1976). Hormonal gradients within the suspensor are also indicated by the gradient in D N A synthesizing activity and endopolyploidy (Nagl, 1974, 1976a). In this connection it should not
W. Nagl and S. K/ihner: Diversification of Plastids
Fig. 2. Optical micrograph of a cell isolated from the cotyledo of a heart-stage embryo. Note the numerous starch grains. Ethanol-acetic acid, phase contrast, x 200, The bar indicates 5 ~tm Fig. 3. Electron micrograph of an amyloplast in the cotyledo of a heart-stage embryo, x 20,000. The bar indicates 1 gm Fig. 4. Part of a chloroplast showing a well-developed granum, as found in the meristematic region of the embryo proper, x 32,000. The bar indicates I gm Fig. 5. Part of an etioplast with prolamellar bodies as seen in cells of the placental haustorium, x 20,000, The bar indicates 1 [am
be i g n o r e d that the n u c l e a r D N A c o n t e n t also affects the n u m b e r a n d size of plastids (Butterfass, 1973) as well as the n u m b e r of lamellae within the g r a n a (Lee a n d Hecht, 1975). The different D N A c o n t e n t s that have been f o u n d in various cells of the e m b r y o a n d s u s p e n s o r of Tropaeo/urn (Nagl, 1976b; Nagl et al., 1976) may, therefore, play some role in the
c o n t r o l of u l t r a s t r u c t u r a l differentiation of the plastids. The f u n c t i o n a l significance of the various ultrastructures, which have been observed in the e m b r y o a n d s u s p e n s o r of Tropaeolurn, is n o t yet u n d e r s t o o d . The following speculations m a y be made, The suspensor cells with leukoplasts are m o r e active in very early
W. Nagl and S. Ktihner: Diversification of Plastids
Fig. 6. Leukoplasts with electron-dense matrix and electron-transparent tubules, as found in the basal cell mass of the embryo-suspensor. • 15,000. The bar indicates 1 pm Fig. 7. Leukoplast in a basal cell mass during autolytic transformation. Note the electron-transparent tubules, x 22,500. The bar indicates 1 pm Fig. 8. Semithin sectioned chloroplast cluster within an elongated cell of the embryo-snspensor. Phase contrast, x 1200. The bar indicates 10 gm Fig. 9. Ultrathin sectioned chloroplast cluster within a cell of the embryo-suspensor. A part of the nucleus can be seen at left. x 15,000. The bar indicates 1 gm
e m b r y o g e n y a n d synthesize m a i n l y lipids, as s h o w n in an earlier i n v e s t i g a t i o n (Nagl, in press). The dense m a t r i x a n d the t u b u l e s o f the l e u k o p l a s t s in the b a s a l cell m a s s a n d the c a r p e l h a u s t o r i u m are c o n s i s t e n t with the a s s u m p t i o n t h a t these plastids p l a y some role in m e t a b o l i t e t r a n s p o r t (cf. C h a p m a n e t a l . , 1975), o r in the secretion o f m a t e r i a l (cf. D u m a s ,
1975; F o u g e r e - R i f o t , 1975), o r in p h y t o h o r m o n e release (cf. E v a n s a n d Smith, 1976). The f u n c t i o n o f s u s p e n s o r plastids in the n o u r i s h m e n t o f the e m b r y o has been e n v i s a g e d for Pisum ( M a r i n o s , 1970), Ipomoea (Ponzi a n d P i z z o l o n g o , 1973), a n d Stellaria ( N e w c o m b a n d F o w k e , 1974). U n u s u a l plastids have also been f o u n d in PhaseoIus ( S c h n e p f a n d N a g l ,
W. Nagl and S. Kfihner: Diversification of Plastids
1970). Biochemical attempts are now under progress in order to elucidate the early differentiation process in Tropaeolum.
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Received 10 July; accepted 2 September 1976