Planta (BerI.) 104, 157-166 (1972) 9 by 8pringer-Verlag 1972

Embryogenesis and Germination in Rye (Secale cereale L.) 1. Fine Structure of the Developing E m b r y o Nell D. Hallam Agricultural Research Council Unit of Developmental Botany, Cambridge, U.K. Received November 2, December 28, 1971

Summary. Cells of the young embryo contain highly differentiated organelles. During maturation and dehydration, complexity is reduced, the many layers of endoplasmic reticulum associated with electron lucent bodies become reduced to a few residual crescents, lipid droplets distributed in the cytoplasm migrate to and become closely appressed to the plasmalemma, mitochondriM cristae are reduced in number and dictyosomes are compacted. Introduction Several workers have reported the changes in fine structure which occur during embryogenesis in a number of species (Jensen, 1965; Jensen and Fisher, 1967; Schultz and Jensen, 1968; Bain and Mercer, 1966; Marinos, 1970; Mollenhauer and Torten, 1971) and others have studied the changes which take place during germination (Berjak, 1968; Berjak and Villiers, 1970; Perner, 1965; u 1970; Durzan et al., 1971; H o m e r and Arnott, 1966). This paper describes the ultrastructmoe and developmerit of the embryo together with the changes that occur in the organelles during the maturation of the grMn of Seeale cereale. I t is the first of a series which follows the ultrastructural and associated biochemicM changes which occur during embryogcnesis and maturation to the dry grain and thence through the early hours of germination.

Materials and Methods At one, two, three and four weeks following anthems, the whole ovary was excised from the floret, the style arms removed and the ovary walls sliced off to facilitate the penetration of fixative and solvents. The seeds were full sized by four weeks, the endosperm being milky and the grain pale yellow. From the mature, dry embryo, lateral root primordia were dissected with their adherent eoleorhiza. Tissues were fixed in 6 % glutaraldehyde in 0.1 )/I sodium cacodylate buffer at pH 7.1 for 2 h. They were dehydrated ttn~ough an ethanol series, transferred to acetone, embedded in TAAB resin (TAAB Laboratories, Reading, U.K.) and polymerized overnight at 60~ C. 11 Planta(Berl.),Bd. 104

Fig. 1

N. D. Hallam: Embryogenesis and Germination in Rye

159

Thin sections were cut and stained either with lead citrate (t~eynolds, 1963) alone for one hour, or with lead citrate followed by saturated uranyl acetate in 50 % ethanol for one hour. Specimens were examined in a Philips EM 300 electron microscope at either 40 or 60 kV. Estimations of the number of mitochondria were derived by counting twenty cells from the appropriate areas of ten embryos. Results

T h r o u g h o u t this s t u d y attention has been confined to root primordia. One week after anthesis, the developing embryo is shaped like a blunt tailed c o m m a and composed of thin walled cells, m a n y of which are in mitosis (Fig. 1). Vacuolate cells of the o v a r y are separated from the e m b r y o b y an osmiophilic cuticle-like layer (Fig. 2) with microtubules visible on both sides of the wall. The cytoplasm contains numerous ribosomes, often in polysomal aggregations (Fig. 9). Dictyosomes, proplastids and mitochondria are abundant, the latter elongated and with m a n y cristae. The well defined rough endoplasmic reticulum appears in short lengths. After two weeks, there is an increase in the n u m b e r of ribosomes and thereafter t h e y are so a b u n d a n t t h a t polysome aggregations arc indistinguishable (Figs. 4 and 5). Numerous plastid-like organelles are present, some resembling enlarged dividing mitochondria or proplastids (Fig. 4), others containing what appears to be starch (Fig. 5). A t this stage the endoplasmic reticulum is extensive, but after three weeks (Fig. 6) it becomes associated with bodies resembling mitochondria in size t h o u g h having an electron lucent, granular appearance. Four weeks after anthesis (Fig. 7) the endoplasmic reticu]um surrounding these organelles is reduced to single circlets, b u t mitochondria still have numerous cristae. W h e n the seed is mature and d e h y d r a t e d (5-6 weeks) small crescents of endoplasmic reticulum only remain associated with the electron lucent bodies (Fig. 8) while a few cristae remain in the mitochondria. E a c h root primordinm in the d r y mature e m b r y o can be divided into four distinct zones, each with a characteristic cell shape: the root cap, with closely packed cells, the presumptive meristem of smaller cuboidal cells and the procortical and pro-stelar areas composed of elongated, horizontally stacked rectangular cells (Fig. 11). The cytoplasm of all cell types is dense and the nuclei, which have prominent nucleoli, contain a b u n d a n t chromatin (c.f. Figs. t and 11).

Fig. 1. Section through the presumptive root tissue of the developing embryo approximately one week after anthesis. One of the cells is seen in polar view during mitosis. The cell wails are thin with numerous plasmadesmata (P), and have large nuclei with diffuse patches of densely staining chromatin. No nucleoli are evident. Note that there are relatively large droplets of lipid (L) in most cells. • 6384 11"

Figs. 2-5

:N. D. Hallam: Embryogenesis and Germination in Rye

161

T h e m i t o e h o n d r i a of m e r i s t e m a t i e cells a l t h o u g h fewer t h a n in o t h e r cell t y p e s c o n t a i n t h e highest n u m b e r of cristae (3 to 5 p e r m i t o e h o n d r i a l section) a n d m a n y m i t o c h o n d r i a contain small osmiophilic bodies : 2 - 3 p e r section of each organelle (Fig. 8). A dense a g g r e g a t i o n of ribosomes a n d p r o - a m y l o p l a s t s are also p r e s e n t in t-he m a t u r e e m b r y o (Fig. 11). The l a t t e r m a y be d e r i v e d from t h e s t a r c h containing organelles seen a t t h e two week p o s t - a n t h e s i s stage. I n t h e m a t u r e e m b r y o , p r o - a m y l o p l a s t s are 2 - 3 times t h e size oi m i t o e h o n d r i a a n d contain a group of between five a n d t e n osmiophilic a g g r e g a t e d particles often with spindle like a t t a c h m e n t s . Organelles similar in a p p e a r a n c e a n d acid p h o s p h a t a s e reaction to t h e lysosomes d e s c r i b e d b y B e r j a k a n d Villiers (1968) are freq u e n t l y seen r a n d o m l y d i s t r i b u t e d within t h e cells of t h e r o o t cap. A r o u n d t h e inside of t h e p l a s m a l e m m a of all cell t y p e s w i t h i n t h e m a t u r e e m b r y o are small osmiophilic lipid bodies (Figs. 10 a n d 11). Similar d r o p l e t s are seen t h r o u g h o u t t h e c y t o p l a s m d u r i n g embryogencsis (Figs. l , 2, 3, 4, a n d 5) b u t t h e y do n o t m o v e to t h e p l a s m a l e m m a u n t i l t h e final stage of d e h y d r a t i o n . A t high m a g n i f i c a t i o n u n d e r o p t i m u m resolution, an

Fig. 2. Junction of the developing seed and the ovary wall one week after anthesis. l~elatively large osmiophilic lipid droplets are present both within the embryo tissue (E) and the vacuolate cells of the ovary wall (0). A cuticle-like boundary separates the two tissue types. • 4 788 Fig. 3. High magnification of an area similar to that in Fig. 2. The mitochondria have numerous cristae; microtubules and lipid bodies are present. On the outside of the cell wall there is a dense osmiophilie cuticle-like layer. • 30780 Fig. 4. Pro-plastid (P) apparently in division, two weeks after anthesis. Lipid bodies of irregular shape are scattered throughout the ribosome-rich cytoplasm. The mitochondria (M) have numerous cristae. • 30780 Fig. 5. Plastid-like bodies containing starch in the embryo 2 weeks after anthesis. • 30 780 Fig. 6. Endoplasmic reticulum forming numerous crescents associated with electron lucent bodies (E) three weeks after anthesis. • 23 940 Fig. 7. Single circlets of endoplasmic reticulum associated with the electron lucent bodies (E) 4 weeks after anthesis. Mitochondria (M) still have numerous cristae. • 30780 Fig. 8. In the mature, dry embryo, electron lucent bodies (E) generally have a well defined crescent of rough endoplasmic reticulum associated with them. The mitochondria (M) have few cristae. • 37 620 Fig. 9. One week after anthesis. Dictyosomes (D) are numerous and active. :No lipid bodies at plasmalemma. • 41040 Fig. 10. Flattened vesicles resembling a dictyosome in the dry embryo. Note that many densely staining lipid bodies are closely appressed to the plasmalemma. • 3i920

Figs. 6-8. Leg. see p. 161

~igs. 9 ~nd 10. Leg. see p. 161

164

N . D . I-lallam:

Fig. l l

Embryogenesis and Germination in Rye

165

electron-dense layer approximately one third of the width of a unit membrane can be seen around the lipid bodies. They are situated along the plasmalemma, with a mean packing frequency of 4.2 per ~xm. In surface view they form a close order array on the plasmalemma, each droplet being laterally compressed by its neighbours. Dictyosomes are a common feature of tissues at the earlier stages of embryogenesis, as might be expected in tissue that is metabolically active (Fig. 9). They are less frequent three weeks after anthesis and in the mature embryo, elongated and twisted stacks of vesicles are the only vestiges of dietyosomes that remain (Fig. 10). In contrast, the nucleolus becomes increasingly well defined at the later stages of dehydration and the ehromatin aggregates into dense masses (e.f. Figs. 1 and 11). Discussion During the early divisions in cmbryogenesis, the cellular organelles show a high degree of structural complexity. As the embryo matures and dehydrates the complexity is reduced, the dry embryo showing simple or attenuated organelles typical of tissue with low metabolic activity. For example, young cells of the rapidly developing embryo at one or two weeks after anthesis have mitochondria with many cristae, dictyosomes with abundant vesicles and ribosomes frequently in polysomal aggregates (Fig. 9). Three weeks after anthesis (Fig. 6) electron lucent bodies are well defined and associated with many concentric layers of endoplasmic retieulum ; later, the endoplasmie retieulum is reduced to single small crescents around the bodies (Figs. 7 and 8). A reduction in protein synthesis occurs as the seed matures for which the decrease in rough endoplasmie retieulum may be part responsible. In the dehydrated mature embryo, mitochondria are simple with few eristae (Fig. 8), the dictyosomes are few and compacted (Figs. 10 and 11) and no polysomes can be discerned (Fig. 8). Lipid bodies are distributed throughout the cytoplasm in early embryogenesis (Figs. 1, 2, 3, and 5), but in the last stages of dehydration they are packed against the plasmalemma, a situation associated with metabolically inactive tissue by Yoo (1970) and Abdul-Baki and Baker (1970). These bodies are not organellcs in the structural sense, since although they appear to be bounded by a single membrane, Frey-Wyssling et al. (1963) have pointed out that an oil

Fig. 11. Section through a cell of the presumptive cortical area of the root primordium of the dry embryo. The nucleus is large and contains a nucleolus (Nu) and deeply staining ehromatin (Cr). Pro-amyloplasts (A), mitochondria (M) and electron lucent bodies (E) are present. Lipid droplets (L) are closely packed along the plasmalemma. Some droplets have aggragated to form larger globules. • 12540

166

N.D. Hallam: Embryogenesis and Germination in Rye

a n d p r o t e i n interface can appear as a n electron dense m e m b r a n e in the electron microscope. The second paper in this series describes a reverse sequence of ultras t r u c t u r a l changes which occurs as the e m b r y o germinates, together with the sequence of biochemical events to which these changes are linked. References Abdul-Baki, A., Baker, J. E. : Changes in respiration and cyanide sensitivity of the barley floret during development and maturation. 1)lant Physiol. 45, 698-702 (1970). Bain, J. M., Mercer, F. V.: Snbcellular organization of the developing cotyledons of Pisum sativum L. Aust. J. biol. Sei. 19, 49-67 (1966). Berjak, P.: A lysosome-like organelle in the root cap of Zea maya. J. Ultrastruct. Res. 27, 233-242 (1968). Berjak, P., Villiers, T. A. : Ageing in plant embryos. I. The establishment of the sequence of development of senescence in the root cap during germination. New Phytol. 69, 929-938 (1970). Durzan, D. J., Mia, A. J., Ramaian, 1). K. : The metabolism and subcellular organization of the jack pine embryo (Pinus banksiana) during germination. Canad. J. Bet. 49, 927-938 (1971). Fisher, D. B.: Cotton embryogenesis: Double fertilization. 1)hytomorphology 17, 261-269 (1967). Frey-Wyssling, A., Grieshaber, E., Mfillethaler, K. : Origin of spherosomes in plant cells. J. Ultrastruet. Res. 8, 506-516 (1963). Morner, H. T., Jr., Arnott, H. J. : A histochemical and ultrastructural study of pre and post germianted Yucca seeds. Bet. Gaz. 127, 48-64 (1966). Jensen, W. A. : The ultrastructure and composition of the egg and central cell of cotton. Amer. J. Bet. 52, 781-797 (1965). Marines, N. G.: Embryogenesis of the Pea (Pisum sativum). I. The cytological environment of the developing embryo. Protoplasma 70, 261-279 (1970). Mollenhauer, H. I-I., Totten, C.: Studies on seeds. II. Origin and degradation of lipid vesicles in pea and bean cotyledons. J. Cell Biol. 48, 395-405 (1971). Perner, E. : Electronenmikroskopische Untersuchungen an Zellen yon Embryonen im Zustand vSlliger Samenruhe. 1. Mitt. : Die zellulare Strukturordnung in der Radicula lufttrockener Samen yon Pisum 8ativum. 1)lanta (Berl.) 65, 334-357 (1965). Reynolds, E. S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-213 (1963). Schulz, Sister l~ichardis, Jensen, W. A. : CapseUa Embryogenesis: The early embryo J. Ultrastruct. Res. 22, 376-392 (1968). Yoo, B. Y. : Ultrastructural changes in cells of pea embryos and radicles during germination. J. Cell Biol. 45, 158-171 (1970). Dr. Neil D. Hallam Agricultural Research Council Unit of Developmental Botany 181A Huntingdon Road Cambridge, U.K. present address Botany Department Monash University Clayton, Vie. 3168, Australia