Planta (Berl.) 79, 346--366 (1968)
Cotton Embryogenesis : The Zygote WILLIAM A. JE~S~N Department of Botany, University of California, Berkeley Received October 25, 1967
Summary. The zygote of cotton (Gossypium hirsutum) remains undivided for approximately 21/2 days following fertilization. The changes which occur during this period can be divided into two stages. During stage 1 the zygote decreases in volume so that its volume becomes one half that of the egg. Correlated with this change a number of Mterations occur in the endoplasmic reticulum (EI~) numerous enlargements form; it becomes closely associated with the plasma membrane; and an internal network of tubes appears in it. The plastids and mitochondria become grouped around the zygote nucleus. The ribosomes form large, helical polysomes which are arranged as shells around the plastids and mitochondria. Starch accumulation and wall formation over the chalazM end of the cell, which begins during the end of stage 1, continues during stage 2. A new set of ribosomes appear in the cytoplasm. These either remain single, or aggregate into small polysomes. The large, helical po]ysomes of stage 1 persist. Ultimately the zygote becomes a highly polarized cell, rich in starch, surrounded by a wall, filled with a tube containing ER, and two types of polysomes, one composed of ribosomes present in the egg and the other of ribosomes produced by the zygote nucleus. Introduction The t r a n s f o r m a t i o n of t h e egg to t h e z y g o t e in c o t t o n is m a r k e d b y a d r a m a t i c series of a l t e r a t i o n s in cell s t r u c t u r e . These changes, m a n y of which are visible with t h e light microscope, are n o t d e s c r i b e d in t h e earlier studies of c o t t o n e m b r y o g e n e s i s (BALLS, 1905 ; Gom~, 1932). B e t w e e n t h e t i m e t h a t t h e s p e r m nucleus e n t e r s t h e egg a n d t h e division of t h e resulting zygote, t h e r e are changes in cell size, v a c u o l a t i o n , p o s i t i o n of t h e e n d o p l a s m i c r e t i c u l u m (ER), f o r m a t i o n a n d d i s t r i b u t i o n of r i b o s o m e s a n d polysomes, a m o u n t of starch, protein, a n d nucleic acids, a n d e x t e n t of t h e wall. This p a p e r is a p r e l i m i n a r y r e p o r t of these changes as o b s e r v e d w i t h t h e light a n d electron microscopes. The o b j e c t of this r e p o r t is to p r e s e n t an overall view of t h e changes listed above. Topics of special i n t e r e s t will be t r e a t e d in g r e a t e r d e t a i l in o t h e r p u b l i c a t i o n s : fusion of t h e g a m e t i c nuclei (J]~SEN a n d F I S R ] ~ , 1968b); t h e t u b e - c o n t a i n i n g E R (J~Ns~N, 1968a); a n d t h e p o l y s o m e s ( J ~ s ~ , 1968b). Materials and Methods Plant Material. Upland cotton plants (Gossypium hirsutum L., seed variety M8948) were grown in a greenhouse in which supplementary fluorescent lights
Cotton Embryogenesis: The Zygote
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provided an 18-hour photoperiod. The seed employed is a double haploid developed by Dr. JAnus MEYEgS, Delta Branch Experimental Station, Stoneville, Mississippi. Flowers were hand-pollinated at 9a.m. each day and the stamens removed. Under these conditions fertilization occurs in about 13--18 hours following pollination, depending on a variety of environmental conditions. Light Microscopy. Ovules were dissected from the flowers at various lengths of time following pollination and frozen in isopentane cooled with liquid nitrogen. They were then dried in a moving-gas-type freeze-dry apparatus (JnNsEN, 1962, p. 100), and parMfin-infiltrated and embedded. Ovules and isolated nucelli were also fixed in cold 3% glutarMdehyde in 0.05 M cacodylate buffer, pH 6.8, for 12 hours. The tissue was then washed, dehydrated, and embedded in Epon. Sections at 1.5--2.0 tz were cut on a PorterBlum ultramierotome. Electron Microscopy. Isolated nueelli were fixed in 3 % glutaraldehyde in 0.05 M eaeodylate buffer, pI-I 6.8, for 12 hours, washed, and postfixed overnight in 2% OsO 4, dehydrated in acetone and Epon-embedded. The 70% acetone step for dehydration eontMned 1% uranyl nitrate, in which the tissue remained overnight. Thin sections were eat on a Porter-Blum ultramicroteme, stained with Reynold's lead citrate, and examined with a Zeiss EM 9 electron microscope. Quantitative Morphological Analyses-Models. Freeze-dried ovules were transeetioned at 5 ~z, and the sections were pressed on slides coated with adhesive (JE~rSEX, 1962, p. 199). Slides were deparaffinized with xylene and cover slips applied using phase-contrast mounting media (Zeiss-L-15). Consecutive sections of the embryo sac were photographed under phase contrast, and prints were made. Cell areas were determined by planimeter measurements of the various cells in the photographs. Plastic models were constructed to better understand the form of the synergids and the egg-zygote. For the models, tracings of the cells were made from the photographs, and these were transferred to a/s-ineh-(ca. 0.5 cm-)thick sheets of plexiglass plastic using carbon paper. The plastic was cut on a jigsaw, the edges polished, nuclei, nucleoli and other cell features were etched in with an engraving tool and colored with india ink. The enlargement was such that the a/s-inch plastic accurately represented a 5-~z-thick section. Therefore the models are accurate with respect to all spatial configurations.
Results General Change in Size and Shape. T h e r e s u l t s of t h e a n a l y s e s of c o n s e c u t i v e 5 ~ s e c t i o n s t h r o u g h t h e egg a n d z y g o t e a r e s h o w n in Fig. 1. T h e s e c t i o n s are n u m b e r e d f r o m t h e m i c r o p y ] a r t i p of t h e e m b r y o sac. T h e t w o s y n e r g i d s o c c u p y t h e e n t i r e a r e a of t h e first f o u r sections, t h e egg or z y g o t e first a p p e a r i n g in t h e f i f t h s e c t i o n . T h e egg is a p e a r - s h a p e d cell, 9 0 - - 1 0 0 ~t in l e n g t h . T h e r e l a t i o n s h i p of t h e egg t o t h e t w o s y n e r g i d s can be s e e n in Fig. 2a. T h e egg, while a p p r o x i m a t e l y t h e s a m e size as t h e s y n e r g i d s , is p l a c e d m o r e c h a l a z M l y in t h e e m b r y o sac a n d t h u s o v e r t o p s t h e s y n e r g i d s . T h e z y g o t e , 24 h o u r s a f t e r p o l l i n a t i o n o r a p p r o x i m a t e l y 8 - - 1 0 h o u r s a f t e r e n t r a n c e of t h e s p e r m n u c l e u s i n t o t h e egg, is h a l f t h e v o l u m e of t h e egg. T h e d e c r e a s e in v o l u m e is f r o m t h e c h a l a z a l e n d of t h e cell. T h e m i c r o p y l a r 3 5 - - 4 0 ~t of t h e z y g o t e are t h e s a m e as t h e y w e r e in t h e egg.
348
W.A. JENSEN:
The spatial relationship of the zygote to the two synergids is shown in Fig. 2b. The models illustrated in Fig. 2a and 2b are the same in scale so that the volume of the egg and zygote can be directly compared. The difference in size and shape of the egg and zygote can also be seen in the electron-microscope photographs shown in Fig. 3. The egg is slightly collapsed on one side, but the section accurately reflects the gross morphology of the cell. The zygote shown in Fig. 3 is 3 days after pollination and is in division. The section of the zygote is almost median for the cell although not for the nuclear region. IlO
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Fig. i. The relation of the form of the egg to that of the average of either three eggs or three zygotes. after pollination or i0 hours after fertilization. The sac are occupied completely by the two synergids
the zygote. Each curve represents The zygotes were fixed 24 hours first four sections of the embryo
(see JENS~g and FIS~E~, 1968a)
From Fig. 3 it is clear that the decrease in by a marked decrease in the size of the central which was spread in a thin layer between the membrane of the egg, accumulates around the
cell size is accompanied vacuole. The cytoplasm, vacuole and the plasma zygote nucleus.
I n order to describe the development of the zygote, the sequence has been divided into a number of stages. These stages and the time periods they occupy following pollination are as follows : The egg (0--14 hours) ; zygote-stage 1 : nuclear fusion and reorganization ( 1 ~ - 3 6 hours); zygotestage 2: maturation (36--65 hours). These periods and times are not absolute and are intended only to form a framework for organizing the data. Egg (0--14 Hours). The structure of the egg in cotton has been described in detail on the basis of permanganate-fixed material ( J ~ s ~ N , 1965). Only a summary of in~portant features of the egg and additional new data collected from Ga-Os-fixed tissue will be presented here.
Cotton Embryogenesis: The Zygote
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The egg is partially surrounded by a wall. The wall is thickest at the micropylar end of the cell and extends approximately one half the length of the egg. There is no wall over the chalazal half of the egg, and the contact between the egg and the synergids Or central cell involves only plasma membranes. The central vacuole is large and the cytoplasm pressed in a thin layer between it and the plasma membrane (Figs. 3, 4, and 5). The greatest
350
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Fig. 3. Median sections of the egg (left) and a 3-day-old zygote (right). The position of the egg nucleus in the upper right hand third of the egg is characteristic although the egg has collapsed slightly. The zygote nucleus is in division. The micropyle is down. Both sections at the same magnification. GA-Os fixation c o n c e n t r a t i o n of t h e c y t o p l a s m is f o u n d a t t h e m i c r o p y l a r end of t h e egg s u r r o u n d i n g t h e nucleus. T h e egg nucleus is u s u a l l y f o u n d in the chalazal t h i r d of t h e egg, One nueleolus is p r e s e n t in t h e egg nucleus. R i b o s o m e s are n u m e r o u s (Figs. 4 a n d 5) a n d are f o u n d b o t h a t t a c h e d to the E R a n d u n a t t a c h e d in t h e g r o u n d c y t o p l a s m . The u n a t t a c h e d
Cotton Embryogenesis: The Zygote
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Fig. 4. Portion of the egg and central cell (CC) of an 0-day embryo sac. Note the random distribution of the ribosomes and lack of association with either the plastid (P) or mitochondrion (M). GA-Os fixation. • 59,000 Fig. 5. Portion of egg a n d central cell (CC) of an 0-day embryo sac. The dictyosome (D) appears relatively inactive, and ribosomes are randomly arranged around it a n d the segment of a plastid (P) present in the section. GA-Os fixation; • 59,000
352
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Fig. 6. A zygote 18 hours after pollination or approximately 4 hours after fertilization. The sperm a n d egg nuclei are not completely fused, b u t only the egg portion is visible in this section. Note the arrangement of the plastids and mitoehondria. GA-Os fixation; • 2,350
Cotton Embryogenesis: The Zygote
353
Fig. 7. A zygote 24 hours after pollination or approximately 10 hours after fertilization. The zygote nucleus is completely formed although only one of the two nucleoli is visible. This zygote is unusual in that the nucleus is at the mieropylar rather than the chalazal end of the cell. The absence of a wall at the chalazal end of the cell is clear in this picture. Portions of the degenerate synergid (DS) and the endosperm (E) are visible. • 1,750 ribosomes show a low degree of aggregation into polysomes. The p l a s t i d s c o n t a i n small s t a r c h grains, a n d t h e m i t o c h o n d r i a m o d e r a t e n u m b e r s of cristae (Fig. 4). The d i c t y o s o m e s a p p e a r n o t to be p r o d u c i n g m a n y vesicles (Fig. 5).
Zygote-Stage 1: Nuclear Fusion-Reorganization (14--36 Hours). The pollen t u b e e n t e r s t h e e m b r y o sac t h r o u g h one of t h e synergids a p p r o x i m a t e l y 14 hours a f t e r pollination (JE~sEN a n d F I s H ~ , 1968a). W i t h i n 1/2 h o u r t h e s p e r m nuc]eus, free of c y t o p l a s m , a p p e a r s in t h e egg and
354
W . A . JE~S~N:
Fig. 8 a n d 8A (for legend see 1o. 355)
Cotton Embryogenesis: The Zygote
355
becomes pressed to the egg nucleus. The details of nuclear fusion are presented in another report (JENsEN and FmHE~, 1968b) and will not be given here. The overall form of the cell is not changed by the entrance of the sperm nucleus. After nuclear fusion begins the cell begins to shrink in size. The zygote in Fig. 6 is 18 hours after pollination and only slightly smaller than the egg. Nuclear fusion is not yet complete (only the egg nucleus is visible in this section), but there is an accumulation of plastids and mitochondria around the nucleus. By 24 hours after pollination the zygote has reached its final, reduced size (Fig. 7). The zygote shown in Fig. 7 is unusual in that the vacuole is at the ehalazal rather than the micropylar end of the cell. This difference does not affect the developmental sequence. Nuclear fusion is complete in this zygote, although only one of the two nucleoli can be seen in this section. As the zygote decreases in size, the mitochondria and plastids of the cell become grouped around the nucleus (Figs. 6 and 7). The ribosomes in turn collect around the plastids and mitochondria (Figs. 8 and 9). The bulk of the ribosomes are now aggregated into polysomes. These polysomes show striking helical arrangements of ribosomes. More than 20 ribosomes m a y be found in a single polysome (Fig. 8A). The ground cytoplasm becomes clear in appearance, and few unaggregated ribosomes can be found. Ribosomes are also seen bound to the ER, but the concentration Of these ribosomes in the cell is not great at this stage. During the period of cell shrinkage an unusual E R is seen to increase in the cell (Fig. 10). This is the tube-containing EI~ which is found in small amounts in the egg (JE~s]~>L 1965). The tubes have a diameter of 300 A and are several microns in length. They can be seen to branch and appear to be intertwined with one another within the E R (Fig. 10). They are not straight but can be seen to bend in irregular patterns. One of the characteristics of the tubes is that they are fixed in KMn04 as well as ha Ga-Os. Occasionally they can be seen to be connected with the EI~. The E R containing tubes has ribosomes bound to it. The tubes can also be seen in the nuclear membrane of the zygote (Fig. 10A), and most segments of the E R seem attached to the nuclear membrane containing the tubes. Other changes can be observed in the Et~ during this period of zygote reorganization. The total amount of the E R appears to increase. Fig. 8. Portion of ~ zygote 24 hours after pollination. The plastids (P) and mitochondria (M) form a shell around the nucleus (N) with the ribosomes, aggregated as polysomes, forming shells around the plastids a n d mitoehondria. Note the clear appearance of the ground cytoplasm. • 9,250 Fig, 8A. A large helical polysome in a zygote 36 hours after pollination and in
stage 2. • 42,250 25a Planta (Berl.), Bd. 79
Fig. 9. A group of plastids (P), mitochondria (3//), and dictyosomes (D) in a zygote 24 hours after pollination. The arrangement of the polysomes around the plastid is particularly clear in the case of plastid where the cut has been tangential to the surface (P*). Starch (S) has accumulated in all of the plastids. • 25,700
W. A. J ~ s E ~ : Cotton Embryogenesis: The Zygote
357
Numerous swellings, m a n y quite large, can be seen in the E R (Fig. 11). Obvious connections of the El{ to the membrane of the central vacuole can be seen. Finally, m a n y strands of E R can be seen to run perpendicular to the plasma membrane and terminate at it (Fig. l l A). Whether the E R actually ends at the membrane of fuses with it is not clear from the present preparations. The dictyosomes appear to shorten, and the outer eisternae become curved (Fig. 11). Numerous small vesicles can be seen associated with the ends of the cisternue (Fig. 11). Starch begins to accumulate in the plastids during this stage although there is no other apparent changes in plustid structure (Fig. 8). The mitochondria are more dense in appearance, and m a n y are considerably longer than any found in the egg.
Zygote-Stage 2: Maturation (36--65 Hours). By the beginning of the second stage the finM form of the zygote has been estubhshed (Fig. 3). The chMuzM end of the cell is occupied by the nucleus surrounded by the bulk of the plastids and mitochondria of the cell. The micropylar end of the cell is occupied by one or more vacuoles and is relatively sparse in numbers of cell organelles. Thus the zygote in its final form is a highly polarized cell. Even in those cases where the nucleus is not at the chMuzM end of the cell as in Fig. 7, the cell is still highly polarized, and most of the cytoplasm is associated with the nucleus. Possibly the most striking development during this second stage of development is the appearance of a new population of ribosomes (Fig. 12). The helical polysomes formed from the egg ribosomes during stage 1 remain surrounding the plustids and mitochondria (Figs. 12 and 13). Now, in addition to the helical polysomes, ]urge numbers of single ribosomes and small polysomes can be found throughout the zygote cytoplasm (Figs. 10c and 13). The number of ribosomes attached to the El{ also increases during this stage. These are eharacteristicMly seen arranged in curved patterns on the surface of the E R (Fig. 14). Thus the total number of ribosomes in the zygote increases sharply during this stage. This increase is reflected in a marked increase in Azure-B stainability of the zygote. The central vacuole occupies much less of the volume of the cell. The swellings in the E R are gone or greatly reduced, and few connections can be seen between the E R and the central vacuole. The general contrast in the ground cytoplasm increases during stage 2. Where the background was almost completely clear in stage 1, it is much darker and more granular in appearance in stage 2. Dictyosomes with numerous vesicles surrounding them occur throughout the zygote. The wall is now complete around the zygote (Fig. 12). This wall is thickest at the micropylar end of the cell and thinnest at the chalazal end. 25b
21anta (Berl.), Bd. 79
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Fig. 10A--C (for legend see p. 359)
Co,ton Embryogenesis: The Zygote
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The p l a s t i d s become filled with starch. N o t only does t h e size of t h e grains increase, b u t t h e n u m b e r of grains p e r p l a s t i d increases (Figs. 15 a n d 16). Otherwise p l a s t i d s t r u c t u r e does n o t change. There seems to be no increase in t h e n u m b e r of plastids. The m i t o c h o n d r i a are longer a n d contain m o r e cristae. The general impression is t h a t t h e n u m b e r of m i t o c h o n d r i a is increasing a n d t h a t t h e increase is b y division of existing m i t o c h o n d r i a . Discussion D r a m a t i c changes occur in t h e z y g o t e b e t w e e n t h e t i m e of its f o r m a t i o n a n d t h e t i m e of its division: t h e cell shrinks to one-half its original v o l u m e ; t h e vacuole decreases in v o l u m e ; a t u b e - c o n t a i n i n g E R is m a d e ; t h e p l a s t i d s are r e l o c a t e d a n d a c c u m u l a t e masses of s t a r c h ; g i a n t p o l y s o m e s are formed, a n d a second g e n e r a t i o n of r i b o s o m e s a p p e a r s . Clearly, these are i m p o r t a n t changes a n d indicate t h a t this is a period of conversion in cell function a n d a critical one in t h e life of the plant. The m a t u r e zygote is a far different cell t h a n t h e egg a n d a p p e a r s to be a cell supplied with t h e m a t e r i a l s a n d i n f o r m a t i o n for r a p i d division. The first of these changes, t h e r e d u c t i o n in cell volume, is in m a n y w a y s t h e m o s t spectacular. W h i l e cell e n l a r g m e n t a n d elongation are
standard features of plant cell development, decrease in cell size is rare. The decrease in volume must result from the reduction of the large central vacuole present in the egg. This reduction is presumably through loss of water from the egg to the surrounding endosperm. While other mechanisms can be proposed, a simple change in osmotic balance between the zygote and the endosperm could explain the onset and termination of this water loss. The endosperm nucleus divides immediately after formation, and there are striking changes in the cytoplasm of the endosperm (compare the central cell cytoplasm of Figs. 4 and 5 with the endosperm cytoplasm of Fig. 12). These changes result in the formation of a dense mass of cytoplasm around the zygote and are closely correlated with the period of z y g o t e shrinkage. T h e wall of t h e egg would p r e s u m a b l y be a f a c t o r in this new equilibrium a n d be i n v o l v e d in d e t e r m i n i n g the precise shape a n d v o l u m e of t h e zygote. The work of RYczxows~:i (1964), who m e a s u r e d osmotic changes in t h e sap of ovules after fertilization, t e n d s to s u p p o r t these suggestions. Fig. 10A--C. The tube containing E R (A). Two tubes in cross section in the membrane of the zygote nucleus (B). Tubes in both cross and longitudinal view in the E R of a zygote 18 hours after pollination (C). Tube-containing EI~ in a zygote 3 days after pollination. Note the change in ribosome content and contrast in the ground cytoplasm between B and C. All preparations GA-Os fixed and • 59,000
360
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:Fig. 11 and l l A (for legend see p, 361)
Cotton Embryogenesis: The Zygote
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During the actual period of reduction both the plasma membrane and the vacuolar membrane must decrease greatly in area. Neither membrane appears wrinkled or thickened during this period, suggesting that a mechanism for membrane resorption is operating. This could take place by the membranes actually breaking down, or it could involve converting them into ER. The relation of the E R to the plasma membrane and the vacuoles suggests some immediate involvement of the EI~ in the reduction mechanism. This could be through the conversion of the plasma and vacuolar membranes into El~. The enlarged form of the El% as well as the fact that the tube containing E R appears to form primarily during this period supports this possibility. Additional support comes from the fact that the tube-containing E R is not present in a zygote which shrinks little during formation, Capsella bursa-pastoris (ScI~Vl~Z and JEss~N, 1968). Regardless of the mechanism of the reduction of the zygote volume the results of this act on the cell are profound. The cytoplasm, which had been spread thinly over the chalazal end of the egg, is now packed around the zygote nucleus. The nucleus is surrounded by a shell of p]astids and mitochondria. The vacuole still occupies the mieropylar end of the cell, and hence the zygote is a highly polarized cell. Even where the nucleus is not at the chalazal end of the zygote but is more laterally placed the result is the same: the cell is polarized with most of the cytoplasm surrounding the nucleus and the vacuole at the micropylar end. This is probably the most important result of the decrease in volume and most meaningful in terms of the further development of the zygote. These conditions determine the plane of division in the zygote and result in the formation of a small terminal cell and a large basal cell. Changes in numbers and patterns of aggregation of zygote ribosomes may be of considerable importance in understanding early embryonic development. Of particular interest are the large, helical polysomes formed during stage 1. These are aggregations of the ribosomes present in the egg. Once formed they are remarkably stable and remain associated with the p]astids and mitochondria through the division of the zygote. Presumably the ~gent responsible for the aggregation is a stable messcnger-RNA produced by either the gametic nuclei or the young zygote
Fig. 11. The relation of the ER to the plasma membrane (PM) and the vacuole in a zygote 24 hours after pollination. The ER is perpendicular to the plasma membrane at the top of the picture. The single arrows point to vacuole-like enlargements of the ER, and the double arrow points to a junction of the E/~ with a vacuole which in other sections can be seen to be part of the central vacuole. The large lipid deposits (L) are characteristic of the zygote. • 9250 Fig. 11 A. An enlarged view of the upper part of Fig. l l. The plasma membrane is curved and membrane is not seen in a conventional cross section view. • 25,700
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12
13 Figs, 12 and 13 (for legends see p. 363)
Cotton Embryogenesis: The Zygote
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nucleus. This will have to be verified experimentally, b u t the possibility offers considerable promise in u n d e r s t a n d i n g some of the early stages of embryo development. The n e x t change in the ribosome c o m p l e m e n t of the zygote is also interesting. The f o r m a t i o n of new ribosomes, ribosomes which are t r u l y
Fig. 14. The relation of the ribosomes to the E R in a zygote 21/2 days after pollination. The number of ribosomes increases markedly, and long curved chains of ribosomes are numerous. • 42,250 the p r o d u c t of the zygote, first occurs after nuclear fusion is complete. These are aggregated into m u c h smaller polysomes t h a t are n o t helical. T h e y are p r o b a b l y involved in protein synthesis in c o n j u n c t i o n with messenger R N A being produced b y the zygote nucleus. Clearly, there is a n a c c u m u l a t i o n of protein a n d R N A in the zygote before division occurs. Fig. 12. A portion of a zygote 3 days after pollination showing the relation of the large polysomes to the plastids (P) and the mitochondria (M). Note the increase in ribosomes and small polysomes as well as the thickness of the wall (W) and the increase in starch (S). The density of the endosperm (E) should be compared to the central cell in Figs. 4 and 5. • 22,750 Fig. 13. An enlarged view of a zygote 3 days after pollination. Both helical polysomes and the second generation ribosomes and small polysomes can be seen. • 42,250
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Figs. 15 and 16 (for legends see p. 365)
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365
Thus the zygote at division contains a mixture of ribosomes and RNA, products of different stages of development and all involved in the growth of the embryo. Physiological and biochemical experiments, which should more precisely elucidate these interactions, could go far in explaining embryonic development. The present observations provide the basis for such experiments in cotton. The present observations also bear on the problem of carbohydrate metabolism in the developing zygote. Two observations, in particular, appear important. The first is the formation of the wall and the related activity of the dictyosomes. The second is the increase in starch in the plastids. As noted earlier (JENsJ~, 1965), the wall of the egg extends only around the micropylar half of the cell. W h y a complete wall is not formed is by no means clear. The thickness of the wall at the micropylar end argues against the possibility that the cell lacks either the materials or the mechanism for synthesizing a complete wall. I n any case, once the zygote has been formed and the reduction in cell volume is complete, a wall is now laid down. The wall synthesis is directly related to increased activity of the dictyosomes. At the same time that the wall is forming starch is being synthesized in the plastids. Some starch is present in the egg (JENsEN, 1965), usually in the form of one or, at most, two relatively small grains per plastic[. After the zygote has entered phase 2 and the plastids are grouped around the nucleus, both the size and the number of the starch grains per plastid increase markedly (Figs. 15 and 16). By the time division occurs considerable starch is present in the cell. Finally, why was the decrease in zygote volume not noted in the earlier work on cotton by BALLS (1905) and Go~E (1932) ? The change can be seen with the light microscope, yet it was not described. I t is possible that the use of certain chemical fixatives (such as FAA and Navashin's), which are known to produce distortion of the cells of the embryo sac, led BALLS and GOaE to conclude that any shrinkage of the zygote was a fixation artifact. I n addition, this was a stage of little interest. Both fertilization and zygote division have proven much more interesting to the plant embryologists than the "resting" zygote. These considerations bear on the question of how wide-spread in angiosperms is the phenomenon of zygote shrinkage. There are obviously
Fig. 15. The nucleus (N) surrounded by plastids containing relatively small numbers of starch grains of a zygote 18 hours after fertilization. • 2,800 Fig. 16. The nuclear region of a zygote 3 days after fertilization showing the large amount of starch present in the plastids. The nucleus is in division, and some of the chromosomes (Ch) can be seen. • 2,800
366
W.A. JEWSEN: Cotton Embryogenesis: The Zygote
m a n y zygotes which are densely cytoplasmic a n d which reduce little, if any, i n volume. Z~A a n d CAPS~LLA are examples. I n CAPSELLA a n d other species there is elongation of the zygote before division. B u t there are also m a n y angiosperms similar to cotton with highly vacuolate eggs b u t whose zygotes are more densely cytoplasmic (see MAHESItWAI~I, 1950, p. 268). There is a n a m a z i n g lack of d a t a on the relation of the size of the egg a n d zygote i n most angiosperms. Clearly, m a n y species need to be r e e x a m i n e d before a n answer can be given to the original question, b u t I predict t h a t the p h e n o m e n o n will be f o u n d to be widespread in the Angiosperms. The research was supported by grants from the National Science Foundation (GB-3460), the National Institutes of Health (5-RO1-CA3656-10), and the Miller Institute for Basic Science at the University of California. I would like to express my appreciation for the help received from Miss MARYAS~TO~,Miss MARIEMIZELLE, and Mrs. PAULASTETLER. References BALLS, W.L.: The sexuality of cotton. Yearbook, Khcdivial Agric. Soc., Cairo 1905, p. 199--222. Go~E, U. R. : Development of the female gametophyte and embryo in cotton. Amer. J. Bot. 19, 795--807 (1932). JE~SEN, W.A. : Botanical histochemistry. San Francisco: Freeman 1962. - - The ultrastructure and composition of the egg and central cell of cotton. Amer. J. Bot. 52, 781--797 (1965). - - Cotton embryogenesis: The tube containing EI~. J. Ultrastruct. Res. (in press) (1968a). - - Cotton embryogenesis : Polysome formation in the zygote. J. Cell Biol. (in press) (19685). --, and D. B. FIS~Eg: Cotton embryogenesis: Entrance and discharge of the pollen tube into the embryo sac. Planta (Berl.) 58, 158--183 (1968). --Cotton embryogenesis: Double fertilization. Phytomorphology (in press) (1968). MA~ESHWARI, P. : An introduction to the embryology of angiosperms. New York: McGraw-Hill 1950. RYCZKOWSKI, M.: Physieo-chemical properties of the central vacuolar sap in developing ovules. In: Pollen, physiology and fertilization (H. F. LINSKENS, ed.), p. 17--25. Amsterdam: North Holland Publ. Co. 1964. Sc•uLz, R., and W.A. JENSEN: Capsella embryogenesis: The egg, zygote, and early embryo. Amer. J. Bot. submitted 1968. WILLIAM A. JENSEN
Department of Botany University of California Berkeley, California 94720, USA
Cotton Embryogenesis : The Zygote WILLIAM A. JE~S~N Department of Botany, University of California, Berkeley Received October 25, 1967
Summary. The zygote of cotton (Gossypium hirsutum) remains undivided for approximately 21/2 days following fertilization. The changes which occur during this period can be divided into two stages. During stage 1 the zygote decreases in volume so that its volume becomes one half that of the egg. Correlated with this change a number of Mterations occur in the endoplasmic reticulum (EI~) numerous enlargements form; it becomes closely associated with the plasma membrane; and an internal network of tubes appears in it. The plastids and mitochondria become grouped around the zygote nucleus. The ribosomes form large, helical polysomes which are arranged as shells around the plastids and mitochondria. Starch accumulation and wall formation over the chalazM end of the cell, which begins during the end of stage 1, continues during stage 2. A new set of ribosomes appear in the cytoplasm. These either remain single, or aggregate into small polysomes. The large, helical po]ysomes of stage 1 persist. Ultimately the zygote becomes a highly polarized cell, rich in starch, surrounded by a wall, filled with a tube containing ER, and two types of polysomes, one composed of ribosomes present in the egg and the other of ribosomes produced by the zygote nucleus. Introduction The t r a n s f o r m a t i o n of t h e egg to t h e z y g o t e in c o t t o n is m a r k e d b y a d r a m a t i c series of a l t e r a t i o n s in cell s t r u c t u r e . These changes, m a n y of which are visible with t h e light microscope, are n o t d e s c r i b e d in t h e earlier studies of c o t t o n e m b r y o g e n e s i s (BALLS, 1905 ; Gom~, 1932). B e t w e e n t h e t i m e t h a t t h e s p e r m nucleus e n t e r s t h e egg a n d t h e division of t h e resulting zygote, t h e r e are changes in cell size, v a c u o l a t i o n , p o s i t i o n of t h e e n d o p l a s m i c r e t i c u l u m (ER), f o r m a t i o n a n d d i s t r i b u t i o n of r i b o s o m e s a n d polysomes, a m o u n t of starch, protein, a n d nucleic acids, a n d e x t e n t of t h e wall. This p a p e r is a p r e l i m i n a r y r e p o r t of these changes as o b s e r v e d w i t h t h e light a n d electron microscopes. The o b j e c t of this r e p o r t is to p r e s e n t an overall view of t h e changes listed above. Topics of special i n t e r e s t will be t r e a t e d in g r e a t e r d e t a i l in o t h e r p u b l i c a t i o n s : fusion of t h e g a m e t i c nuclei (J]~SEN a n d F I S R ] ~ , 1968b); t h e t u b e - c o n t a i n i n g E R (J~Ns~N, 1968a); a n d t h e p o l y s o m e s ( J ~ s ~ , 1968b). Materials and Methods Plant Material. Upland cotton plants (Gossypium hirsutum L., seed variety M8948) were grown in a greenhouse in which supplementary fluorescent lights
Cotton Embryogenesis: The Zygote
347
provided an 18-hour photoperiod. The seed employed is a double haploid developed by Dr. JAnus MEYEgS, Delta Branch Experimental Station, Stoneville, Mississippi. Flowers were hand-pollinated at 9a.m. each day and the stamens removed. Under these conditions fertilization occurs in about 13--18 hours following pollination, depending on a variety of environmental conditions. Light Microscopy. Ovules were dissected from the flowers at various lengths of time following pollination and frozen in isopentane cooled with liquid nitrogen. They were then dried in a moving-gas-type freeze-dry apparatus (JnNsEN, 1962, p. 100), and parMfin-infiltrated and embedded. Ovules and isolated nucelli were also fixed in cold 3% glutarMdehyde in 0.05 M cacodylate buffer, pH 6.8, for 12 hours. The tissue was then washed, dehydrated, and embedded in Epon. Sections at 1.5--2.0 tz were cut on a PorterBlum ultramierotome. Electron Microscopy. Isolated nueelli were fixed in 3 % glutaraldehyde in 0.05 M eaeodylate buffer, pI-I 6.8, for 12 hours, washed, and postfixed overnight in 2% OsO 4, dehydrated in acetone and Epon-embedded. The 70% acetone step for dehydration eontMned 1% uranyl nitrate, in which the tissue remained overnight. Thin sections were eat on a Porter-Blum ultramicroteme, stained with Reynold's lead citrate, and examined with a Zeiss EM 9 electron microscope. Quantitative Morphological Analyses-Models. Freeze-dried ovules were transeetioned at 5 ~z, and the sections were pressed on slides coated with adhesive (JE~rSEX, 1962, p. 199). Slides were deparaffinized with xylene and cover slips applied using phase-contrast mounting media (Zeiss-L-15). Consecutive sections of the embryo sac were photographed under phase contrast, and prints were made. Cell areas were determined by planimeter measurements of the various cells in the photographs. Plastic models were constructed to better understand the form of the synergids and the egg-zygote. For the models, tracings of the cells were made from the photographs, and these were transferred to a/s-ineh-(ca. 0.5 cm-)thick sheets of plexiglass plastic using carbon paper. The plastic was cut on a jigsaw, the edges polished, nuclei, nucleoli and other cell features were etched in with an engraving tool and colored with india ink. The enlargement was such that the a/s-inch plastic accurately represented a 5-~z-thick section. Therefore the models are accurate with respect to all spatial configurations.
Results General Change in Size and Shape. T h e r e s u l t s of t h e a n a l y s e s of c o n s e c u t i v e 5 ~ s e c t i o n s t h r o u g h t h e egg a n d z y g o t e a r e s h o w n in Fig. 1. T h e s e c t i o n s are n u m b e r e d f r o m t h e m i c r o p y ] a r t i p of t h e e m b r y o sac. T h e t w o s y n e r g i d s o c c u p y t h e e n t i r e a r e a of t h e first f o u r sections, t h e egg or z y g o t e first a p p e a r i n g in t h e f i f t h s e c t i o n . T h e egg is a p e a r - s h a p e d cell, 9 0 - - 1 0 0 ~t in l e n g t h . T h e r e l a t i o n s h i p of t h e egg t o t h e t w o s y n e r g i d s can be s e e n in Fig. 2a. T h e egg, while a p p r o x i m a t e l y t h e s a m e size as t h e s y n e r g i d s , is p l a c e d m o r e c h a l a z M l y in t h e e m b r y o sac a n d t h u s o v e r t o p s t h e s y n e r g i d s . T h e z y g o t e , 24 h o u r s a f t e r p o l l i n a t i o n o r a p p r o x i m a t e l y 8 - - 1 0 h o u r s a f t e r e n t r a n c e of t h e s p e r m n u c l e u s i n t o t h e egg, is h a l f t h e v o l u m e of t h e egg. T h e d e c r e a s e in v o l u m e is f r o m t h e c h a l a z a l e n d of t h e cell. T h e m i c r o p y l a r 3 5 - - 4 0 ~t of t h e z y g o t e are t h e s a m e as t h e y w e r e in t h e egg.
348
W.A. JENSEN:
The spatial relationship of the zygote to the two synergids is shown in Fig. 2b. The models illustrated in Fig. 2a and 2b are the same in scale so that the volume of the egg and zygote can be directly compared. The difference in size and shape of the egg and zygote can also be seen in the electron-microscope photographs shown in Fig. 3. The egg is slightly collapsed on one side, but the section accurately reflects the gross morphology of the cell. The zygote shown in Fig. 3 is 3 days after pollination and is in division. The section of the zygote is almost median for the cell although not for the nuclear region. IlO
u
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Fig. i. The relation of the form of the egg to that of the average of either three eggs or three zygotes. after pollination or i0 hours after fertilization. The sac are occupied completely by the two synergids
the zygote. Each curve represents The zygotes were fixed 24 hours first four sections of the embryo
(see JENS~g and FIS~E~, 1968a)
From Fig. 3 it is clear that the decrease in by a marked decrease in the size of the central which was spread in a thin layer between the membrane of the egg, accumulates around the
cell size is accompanied vacuole. The cytoplasm, vacuole and the plasma zygote nucleus.
I n order to describe the development of the zygote, the sequence has been divided into a number of stages. These stages and the time periods they occupy following pollination are as follows : The egg (0--14 hours) ; zygote-stage 1 : nuclear fusion and reorganization ( 1 ~ - 3 6 hours); zygotestage 2: maturation (36--65 hours). These periods and times are not absolute and are intended only to form a framework for organizing the data. Egg (0--14 Hours). The structure of the egg in cotton has been described in detail on the basis of permanganate-fixed material ( J ~ s ~ N , 1965). Only a summary of in~portant features of the egg and additional new data collected from Ga-Os-fixed tissue will be presented here.
Cotton Embryogenesis: The Zygote
349 o
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The egg is partially surrounded by a wall. The wall is thickest at the micropylar end of the cell and extends approximately one half the length of the egg. There is no wall over the chalazal half of the egg, and the contact between the egg and the synergids Or central cell involves only plasma membranes. The central vacuole is large and the cytoplasm pressed in a thin layer between it and the plasma membrane (Figs. 3, 4, and 5). The greatest
350
W.A. J ~ s ~ :
Fig. 3. Median sections of the egg (left) and a 3-day-old zygote (right). The position of the egg nucleus in the upper right hand third of the egg is characteristic although the egg has collapsed slightly. The zygote nucleus is in division. The micropyle is down. Both sections at the same magnification. GA-Os fixation c o n c e n t r a t i o n of t h e c y t o p l a s m is f o u n d a t t h e m i c r o p y l a r end of t h e egg s u r r o u n d i n g t h e nucleus. T h e egg nucleus is u s u a l l y f o u n d in the chalazal t h i r d of t h e egg, One nueleolus is p r e s e n t in t h e egg nucleus. R i b o s o m e s are n u m e r o u s (Figs. 4 a n d 5) a n d are f o u n d b o t h a t t a c h e d to the E R a n d u n a t t a c h e d in t h e g r o u n d c y t o p l a s m . The u n a t t a c h e d
Cotton Embryogenesis: The Zygote
351
Fig. 4. Portion of the egg and central cell (CC) of an 0-day embryo sac. Note the random distribution of the ribosomes and lack of association with either the plastid (P) or mitochondrion (M). GA-Os fixation. • 59,000 Fig. 5. Portion of egg a n d central cell (CC) of an 0-day embryo sac. The dictyosome (D) appears relatively inactive, and ribosomes are randomly arranged around it a n d the segment of a plastid (P) present in the section. GA-Os fixation; • 59,000
352
W . A . JENSEN:
Fig. 6. A zygote 18 hours after pollination or approximately 4 hours after fertilization. The sperm a n d egg nuclei are not completely fused, b u t only the egg portion is visible in this section. Note the arrangement of the plastids and mitoehondria. GA-Os fixation; • 2,350
Cotton Embryogenesis: The Zygote
353
Fig. 7. A zygote 24 hours after pollination or approximately 10 hours after fertilization. The zygote nucleus is completely formed although only one of the two nucleoli is visible. This zygote is unusual in that the nucleus is at the mieropylar rather than the chalazal end of the cell. The absence of a wall at the chalazal end of the cell is clear in this picture. Portions of the degenerate synergid (DS) and the endosperm (E) are visible. • 1,750 ribosomes show a low degree of aggregation into polysomes. The p l a s t i d s c o n t a i n small s t a r c h grains, a n d t h e m i t o c h o n d r i a m o d e r a t e n u m b e r s of cristae (Fig. 4). The d i c t y o s o m e s a p p e a r n o t to be p r o d u c i n g m a n y vesicles (Fig. 5).
Zygote-Stage 1: Nuclear Fusion-Reorganization (14--36 Hours). The pollen t u b e e n t e r s t h e e m b r y o sac t h r o u g h one of t h e synergids a p p r o x i m a t e l y 14 hours a f t e r pollination (JE~sEN a n d F I s H ~ , 1968a). W i t h i n 1/2 h o u r t h e s p e r m nuc]eus, free of c y t o p l a s m , a p p e a r s in t h e egg and
354
W . A . JE~S~N:
Fig. 8 a n d 8A (for legend see 1o. 355)
Cotton Embryogenesis: The Zygote
355
becomes pressed to the egg nucleus. The details of nuclear fusion are presented in another report (JENsEN and FmHE~, 1968b) and will not be given here. The overall form of the cell is not changed by the entrance of the sperm nucleus. After nuclear fusion begins the cell begins to shrink in size. The zygote in Fig. 6 is 18 hours after pollination and only slightly smaller than the egg. Nuclear fusion is not yet complete (only the egg nucleus is visible in this section), but there is an accumulation of plastids and mitochondria around the nucleus. By 24 hours after pollination the zygote has reached its final, reduced size (Fig. 7). The zygote shown in Fig. 7 is unusual in that the vacuole is at the ehalazal rather than the micropylar end of the cell. This difference does not affect the developmental sequence. Nuclear fusion is complete in this zygote, although only one of the two nucleoli can be seen in this section. As the zygote decreases in size, the mitochondria and plastids of the cell become grouped around the nucleus (Figs. 6 and 7). The ribosomes in turn collect around the plastids and mitochondria (Figs. 8 and 9). The bulk of the ribosomes are now aggregated into polysomes. These polysomes show striking helical arrangements of ribosomes. More than 20 ribosomes m a y be found in a single polysome (Fig. 8A). The ground cytoplasm becomes clear in appearance, and few unaggregated ribosomes can be found. Ribosomes are also seen bound to the ER, but the concentration Of these ribosomes in the cell is not great at this stage. During the period of cell shrinkage an unusual E R is seen to increase in the cell (Fig. 10). This is the tube-containing EI~ which is found in small amounts in the egg (JE~s]~>L 1965). The tubes have a diameter of 300 A and are several microns in length. They can be seen to branch and appear to be intertwined with one another within the E R (Fig. 10). They are not straight but can be seen to bend in irregular patterns. One of the characteristics of the tubes is that they are fixed in KMn04 as well as ha Ga-Os. Occasionally they can be seen to be connected with the EI~. The E R containing tubes has ribosomes bound to it. The tubes can also be seen in the nuclear membrane of the zygote (Fig. 10A), and most segments of the E R seem attached to the nuclear membrane containing the tubes. Other changes can be observed in the Et~ during this period of zygote reorganization. The total amount of the E R appears to increase. Fig. 8. Portion of ~ zygote 24 hours after pollination. The plastids (P) and mitochondria (M) form a shell around the nucleus (N) with the ribosomes, aggregated as polysomes, forming shells around the plastids a n d mitoehondria. Note the clear appearance of the ground cytoplasm. • 9,250 Fig, 8A. A large helical polysome in a zygote 36 hours after pollination and in
stage 2. • 42,250 25a Planta (Berl.), Bd. 79
Fig. 9. A group of plastids (P), mitochondria (3//), and dictyosomes (D) in a zygote 24 hours after pollination. The arrangement of the polysomes around the plastid is particularly clear in the case of plastid where the cut has been tangential to the surface (P*). Starch (S) has accumulated in all of the plastids. • 25,700
W. A. J ~ s E ~ : Cotton Embryogenesis: The Zygote
357
Numerous swellings, m a n y quite large, can be seen in the E R (Fig. 11). Obvious connections of the El{ to the membrane of the central vacuole can be seen. Finally, m a n y strands of E R can be seen to run perpendicular to the plasma membrane and terminate at it (Fig. l l A). Whether the E R actually ends at the membrane of fuses with it is not clear from the present preparations. The dictyosomes appear to shorten, and the outer eisternae become curved (Fig. 11). Numerous small vesicles can be seen associated with the ends of the cisternue (Fig. 11). Starch begins to accumulate in the plastids during this stage although there is no other apparent changes in plustid structure (Fig. 8). The mitochondria are more dense in appearance, and m a n y are considerably longer than any found in the egg.
Zygote-Stage 2: Maturation (36--65 Hours). By the beginning of the second stage the finM form of the zygote has been estubhshed (Fig. 3). The chMuzM end of the cell is occupied by the nucleus surrounded by the bulk of the plastids and mitochondria of the cell. The micropylar end of the cell is occupied by one or more vacuoles and is relatively sparse in numbers of cell organelles. Thus the zygote in its final form is a highly polarized cell. Even in those cases where the nucleus is not at the chMuzM end of the cell as in Fig. 7, the cell is still highly polarized, and most of the cytoplasm is associated with the nucleus. Possibly the most striking development during this second stage of development is the appearance of a new population of ribosomes (Fig. 12). The helical polysomes formed from the egg ribosomes during stage 1 remain surrounding the plustids and mitochondria (Figs. 12 and 13). Now, in addition to the helical polysomes, ]urge numbers of single ribosomes and small polysomes can be found throughout the zygote cytoplasm (Figs. 10c and 13). The number of ribosomes attached to the El{ also increases during this stage. These are eharacteristicMly seen arranged in curved patterns on the surface of the E R (Fig. 14). Thus the total number of ribosomes in the zygote increases sharply during this stage. This increase is reflected in a marked increase in Azure-B stainability of the zygote. The central vacuole occupies much less of the volume of the cell. The swellings in the E R are gone or greatly reduced, and few connections can be seen between the E R and the central vacuole. The general contrast in the ground cytoplasm increases during stage 2. Where the background was almost completely clear in stage 1, it is much darker and more granular in appearance in stage 2. Dictyosomes with numerous vesicles surrounding them occur throughout the zygote. The wall is now complete around the zygote (Fig. 12). This wall is thickest at the micropylar end of the cell and thinnest at the chalazal end. 25b
21anta (Berl.), Bd. 79
358
W . A . JE~sE~:
Fig. 10A--C (for legend see p. 359)
Co,ton Embryogenesis: The Zygote
359
The p l a s t i d s become filled with starch. N o t only does t h e size of t h e grains increase, b u t t h e n u m b e r of grains p e r p l a s t i d increases (Figs. 15 a n d 16). Otherwise p l a s t i d s t r u c t u r e does n o t change. There seems to be no increase in t h e n u m b e r of plastids. The m i t o c h o n d r i a are longer a n d contain m o r e cristae. The general impression is t h a t t h e n u m b e r of m i t o c h o n d r i a is increasing a n d t h a t t h e increase is b y division of existing m i t o c h o n d r i a . Discussion D r a m a t i c changes occur in t h e z y g o t e b e t w e e n t h e t i m e of its f o r m a t i o n a n d t h e t i m e of its division: t h e cell shrinks to one-half its original v o l u m e ; t h e vacuole decreases in v o l u m e ; a t u b e - c o n t a i n i n g E R is m a d e ; t h e p l a s t i d s are r e l o c a t e d a n d a c c u m u l a t e masses of s t a r c h ; g i a n t p o l y s o m e s are formed, a n d a second g e n e r a t i o n of r i b o s o m e s a p p e a r s . Clearly, these are i m p o r t a n t changes a n d indicate t h a t this is a period of conversion in cell function a n d a critical one in t h e life of the plant. The m a t u r e zygote is a far different cell t h a n t h e egg a n d a p p e a r s to be a cell supplied with t h e m a t e r i a l s a n d i n f o r m a t i o n for r a p i d division. The first of these changes, t h e r e d u c t i o n in cell volume, is in m a n y w a y s t h e m o s t spectacular. W h i l e cell e n l a r g m e n t a n d elongation are
standard features of plant cell development, decrease in cell size is rare. The decrease in volume must result from the reduction of the large central vacuole present in the egg. This reduction is presumably through loss of water from the egg to the surrounding endosperm. While other mechanisms can be proposed, a simple change in osmotic balance between the zygote and the endosperm could explain the onset and termination of this water loss. The endosperm nucleus divides immediately after formation, and there are striking changes in the cytoplasm of the endosperm (compare the central cell cytoplasm of Figs. 4 and 5 with the endosperm cytoplasm of Fig. 12). These changes result in the formation of a dense mass of cytoplasm around the zygote and are closely correlated with the period of z y g o t e shrinkage. T h e wall of t h e egg would p r e s u m a b l y be a f a c t o r in this new equilibrium a n d be i n v o l v e d in d e t e r m i n i n g the precise shape a n d v o l u m e of t h e zygote. The work of RYczxows~:i (1964), who m e a s u r e d osmotic changes in t h e sap of ovules after fertilization, t e n d s to s u p p o r t these suggestions. Fig. 10A--C. The tube containing E R (A). Two tubes in cross section in the membrane of the zygote nucleus (B). Tubes in both cross and longitudinal view in the E R of a zygote 18 hours after pollination (C). Tube-containing EI~ in a zygote 3 days after pollination. Note the change in ribosome content and contrast in the ground cytoplasm between B and C. All preparations GA-Os fixed and • 59,000
360
W. A, J ~ s ~ :
:Fig. 11 and l l A (for legend see p, 361)
Cotton Embryogenesis: The Zygote
361
During the actual period of reduction both the plasma membrane and the vacuolar membrane must decrease greatly in area. Neither membrane appears wrinkled or thickened during this period, suggesting that a mechanism for membrane resorption is operating. This could take place by the membranes actually breaking down, or it could involve converting them into ER. The relation of the E R to the plasma membrane and the vacuoles suggests some immediate involvement of the EI~ in the reduction mechanism. This could be through the conversion of the plasma and vacuolar membranes into El~. The enlarged form of the El% as well as the fact that the tube containing E R appears to form primarily during this period supports this possibility. Additional support comes from the fact that the tube-containing E R is not present in a zygote which shrinks little during formation, Capsella bursa-pastoris (ScI~Vl~Z and JEss~N, 1968). Regardless of the mechanism of the reduction of the zygote volume the results of this act on the cell are profound. The cytoplasm, which had been spread thinly over the chalazal end of the egg, is now packed around the zygote nucleus. The nucleus is surrounded by a shell of p]astids and mitochondria. The vacuole still occupies the mieropylar end of the cell, and hence the zygote is a highly polarized cell. Even where the nucleus is not at the chalazal end of the zygote but is more laterally placed the result is the same: the cell is polarized with most of the cytoplasm surrounding the nucleus and the vacuole at the micropylar end. This is probably the most important result of the decrease in volume and most meaningful in terms of the further development of the zygote. These conditions determine the plane of division in the zygote and result in the formation of a small terminal cell and a large basal cell. Changes in numbers and patterns of aggregation of zygote ribosomes may be of considerable importance in understanding early embryonic development. Of particular interest are the large, helical polysomes formed during stage 1. These are aggregations of the ribosomes present in the egg. Once formed they are remarkably stable and remain associated with the p]astids and mitochondria through the division of the zygote. Presumably the ~gent responsible for the aggregation is a stable messcnger-RNA produced by either the gametic nuclei or the young zygote
Fig. 11. The relation of the ER to the plasma membrane (PM) and the vacuole in a zygote 24 hours after pollination. The ER is perpendicular to the plasma membrane at the top of the picture. The single arrows point to vacuole-like enlargements of the ER, and the double arrow points to a junction of the E/~ with a vacuole which in other sections can be seen to be part of the central vacuole. The large lipid deposits (L) are characteristic of the zygote. • 9250 Fig. 11 A. An enlarged view of the upper part of Fig. l l. The plasma membrane is curved and membrane is not seen in a conventional cross section view. • 25,700
362
W.A.J~sEN:
12
13 Figs, 12 and 13 (for legends see p. 363)
Cotton Embryogenesis: The Zygote
363
nucleus. This will have to be verified experimentally, b u t the possibility offers considerable promise in u n d e r s t a n d i n g some of the early stages of embryo development. The n e x t change in the ribosome c o m p l e m e n t of the zygote is also interesting. The f o r m a t i o n of new ribosomes, ribosomes which are t r u l y
Fig. 14. The relation of the ribosomes to the E R in a zygote 21/2 days after pollination. The number of ribosomes increases markedly, and long curved chains of ribosomes are numerous. • 42,250 the p r o d u c t of the zygote, first occurs after nuclear fusion is complete. These are aggregated into m u c h smaller polysomes t h a t are n o t helical. T h e y are p r o b a b l y involved in protein synthesis in c o n j u n c t i o n with messenger R N A being produced b y the zygote nucleus. Clearly, there is a n a c c u m u l a t i o n of protein a n d R N A in the zygote before division occurs. Fig. 12. A portion of a zygote 3 days after pollination showing the relation of the large polysomes to the plastids (P) and the mitochondria (M). Note the increase in ribosomes and small polysomes as well as the thickness of the wall (W) and the increase in starch (S). The density of the endosperm (E) should be compared to the central cell in Figs. 4 and 5. • 22,750 Fig. 13. An enlarged view of a zygote 3 days after pollination. Both helical polysomes and the second generation ribosomes and small polysomes can be seen. • 42,250
364
W . A . JENSEI~:
Figs. 15 and 16 (for legends see p. 365)
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365
Thus the zygote at division contains a mixture of ribosomes and RNA, products of different stages of development and all involved in the growth of the embryo. Physiological and biochemical experiments, which should more precisely elucidate these interactions, could go far in explaining embryonic development. The present observations provide the basis for such experiments in cotton. The present observations also bear on the problem of carbohydrate metabolism in the developing zygote. Two observations, in particular, appear important. The first is the formation of the wall and the related activity of the dictyosomes. The second is the increase in starch in the plastids. As noted earlier (JENsJ~, 1965), the wall of the egg extends only around the micropylar half of the cell. W h y a complete wall is not formed is by no means clear. The thickness of the wall at the micropylar end argues against the possibility that the cell lacks either the materials or the mechanism for synthesizing a complete wall. I n any case, once the zygote has been formed and the reduction in cell volume is complete, a wall is now laid down. The wall synthesis is directly related to increased activity of the dictyosomes. At the same time that the wall is forming starch is being synthesized in the plastids. Some starch is present in the egg (JENsEN, 1965), usually in the form of one or, at most, two relatively small grains per plastic[. After the zygote has entered phase 2 and the plastids are grouped around the nucleus, both the size and the number of the starch grains per plastid increase markedly (Figs. 15 and 16). By the time division occurs considerable starch is present in the cell. Finally, why was the decrease in zygote volume not noted in the earlier work on cotton by BALLS (1905) and Go~E (1932) ? The change can be seen with the light microscope, yet it was not described. I t is possible that the use of certain chemical fixatives (such as FAA and Navashin's), which are known to produce distortion of the cells of the embryo sac, led BALLS and GOaE to conclude that any shrinkage of the zygote was a fixation artifact. I n addition, this was a stage of little interest. Both fertilization and zygote division have proven much more interesting to the plant embryologists than the "resting" zygote. These considerations bear on the question of how wide-spread in angiosperms is the phenomenon of zygote shrinkage. There are obviously
Fig. 15. The nucleus (N) surrounded by plastids containing relatively small numbers of starch grains of a zygote 18 hours after fertilization. • 2,800 Fig. 16. The nuclear region of a zygote 3 days after fertilization showing the large amount of starch present in the plastids. The nucleus is in division, and some of the chromosomes (Ch) can be seen. • 2,800
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W.A. JEWSEN: Cotton Embryogenesis: The Zygote
m a n y zygotes which are densely cytoplasmic a n d which reduce little, if any, i n volume. Z~A a n d CAPS~LLA are examples. I n CAPSELLA a n d other species there is elongation of the zygote before division. B u t there are also m a n y angiosperms similar to cotton with highly vacuolate eggs b u t whose zygotes are more densely cytoplasmic (see MAHESItWAI~I, 1950, p. 268). There is a n a m a z i n g lack of d a t a on the relation of the size of the egg a n d zygote i n most angiosperms. Clearly, m a n y species need to be r e e x a m i n e d before a n answer can be given to the original question, b u t I predict t h a t the p h e n o m e n o n will be f o u n d to be widespread in the Angiosperms. The research was supported by grants from the National Science Foundation (GB-3460), the National Institutes of Health (5-RO1-CA3656-10), and the Miller Institute for Basic Science at the University of California. I would like to express my appreciation for the help received from Miss MARYAS~TO~,Miss MARIEMIZELLE, and Mrs. PAULASTETLER. References BALLS, W.L.: The sexuality of cotton. Yearbook, Khcdivial Agric. Soc., Cairo 1905, p. 199--222. Go~E, U. R. : Development of the female gametophyte and embryo in cotton. Amer. J. Bot. 19, 795--807 (1932). JE~SEN, W.A. : Botanical histochemistry. San Francisco: Freeman 1962. - - The ultrastructure and composition of the egg and central cell of cotton. Amer. J. Bot. 52, 781--797 (1965). - - Cotton embryogenesis: The tube containing EI~. J. Ultrastruct. Res. (in press) (1968a). - - Cotton embryogenesis : Polysome formation in the zygote. J. Cell Biol. (in press) (19685). --, and D. B. FIS~Eg: Cotton embryogenesis: Entrance and discharge of the pollen tube into the embryo sac. Planta (Berl.) 58, 158--183 (1968). --Cotton embryogenesis: Double fertilization. Phytomorphology (in press) (1968). MA~ESHWARI, P. : An introduction to the embryology of angiosperms. New York: McGraw-Hill 1950. RYCZKOWSKI, M.: Physieo-chemical properties of the central vacuolar sap in developing ovules. In: Pollen, physiology and fertilization (H. F. LINSKENS, ed.), p. 17--25. Amsterdam: North Holland Publ. Co. 1964. Sc•uLz, R., and W.A. JENSEN: Capsella embryogenesis: The egg, zygote, and early embryo. Amer. J. Bot. submitted 1968. WILLIAM A. JENSEN
Department of Botany University of California Berkeley, California 94720, USA
