Planta (Berl.) 84, 122--133 (1969)

Cotton Embryogenesis : The Identification, as Nuclei, of the X-Bodies in the Degenerated Synergid DONALD B. FIS~Ea and WILLIA~ A. JEZ~SE~ Department of Botany, University of California, Berkeley Received September 16, 1968

Summary. The "x-bodies" present in the degenerated synergid of cotton were shown to contain DNA by specific staining with Azure B, the Feulgen procedure, and by labelling with 3H-actinomycin D. They are identified on a morphological basis us the degenerated vegetative nucleus of the pollen tube which is always found in the degenerated synergid tip, and as the degenerated synergid nucleus, which is found about halfway up the degenerated synergid near the side towards the central cell. The positions of these nuclei and of some other structures have been used to construct a tentative description of some of the events occurring during pollen-tube discharge and movement of the sperm through the synergid. Some observations which may indicate some of the factors involved in interaction of the synergid ~nd pollen tube cytoplasm are discussed. Introduction Studies of fertilization in Angiosperms have frequently shown the presence of darkly staining structures associated with material discharged from the pollen tube or in the pollen tube itself. These were called x-bodies b y NAVASCHrN and have been interpreted in various ways b y different authors, usually as degenerate nuclei of the synergid, the nucellar cells, supernumerary sperm cells, or the vegetative nucleus of the pollen tube. Occasionally t h e y have been described as the remains of the cytoplasmic sheaths from the sperms (for a review of the literature concerning x-bodies see MAHES~WAnI, 1950). I n an earlier s t u d y of the pollen tube entrance and discharge in cotton ( J ~ s E N and F~SHE~, 1968a) we tentatively described the x-bodies as the remains of the male cytoplasm. This was based largely on electron micrographs of two obviously cytoplasmic structures, in a single degenerated synergid, in positions frequently occupied b y x-bodies. I n all other cases, the x-bodies were obviously degenerate and were not definitely identifiable, on a morphological basis, as either nuclear or cytoplasmic. Since the two

Cotton Embryogenesis: The X-Boclies

123

cytoplasmic bodies were similar in size, shape and position to the x-bodies, it was t h o u g h t the x-bodies were a later stage of the former structures. The fact t h a t we could not account for the synergid or vegetative nuclei after pollen tube discharge constituted an obvious gap in our knowledge concerning pollen tube discharge, and several approaches t o w a r d locating t h e m were taken. Since the morphological characters of practically all structures of ~he synergid a n d pollen-tube contents are profoundly altered after pollen-tube discharge, the most useful approach seemed to be the localization of D N A in the degenerated synergid. However, the unusual staining reactions found in the degenerated synergid make interpretations of reactions ambiguous. For instance, the entfi~e synergid, including the x-bodies, stain with Schiff reagent alone, thus making the interpretation of a Feulgen reaction uncertain. For this reason, D N A was localized most reliably b y t r e a t m e n t of sections with tritiated actinomycin D followed by a u t o r a d i o g r a p h y (EBsTEL~, 1967; CA~ARGO and PLaVT, 1967). More conventional staining procedures were also employed; all procedures agreed in their localization of D N A in the x-bodies. Materials and Methods Upland cotton plants (Gossypium hirsutum L., seed cultivar )[8948) were grown as described previously (JENSENand FISKER, 1968a). For localization of DNA by use of tritiated aetinomycin D, the nucellus was dissected from cotton ovules 24 hours after pollination, quick-frozen in isopentane cooled by liquid nitrogen, freeze-substituted in acetone, and embedded in paraffin (see JENSEN, 1962). Sections 3 y. thick were placed on slides coated with gelatinchrome alum adhesive (JE~sE~, 1962) and deparaffinized. A drop containing 10 ix Cml-1 3H-actinomycin D in water (Schwarz BioI~esearch, Inc.; specific activity 2.97 C mmole-1)was placed on the section. After 1 hour the slide was washed thoroughly in water, dehydrated in 10 rain changes of an alcohol series and air-dried after reaching 95% ethanol. The sections were coated with L-4 emulsion (CA1r and VA~ TU]3ERGEN, 1962), exposed for 20 days, and developed 4 rain in D--19 developer. Controls included treatment with deoxyribonuclease (I hour at 25~ in 1 nag Worthington RNase-free DNase/1.0 ml 0.1 M acetate buffer, pH 5.0, containing 0.005 1HMgSOa) and extraction with hot 0.5 M perchlorie acid (1 hour at 70~ DNA was also localized by Azure-B staining of the sections used for the autoradiography and by Feulgen-staining of h'eeze-substituted, Epon-embedded sections. D~ta on the position of x-bodies were taken from glutaraldehyde-fixed, Eponembedded tissue from our earlier work (JE~sE~ and F~sI~E~, 1968a) and from nucelli which had been freeze-substituted and embedded in Epon. Serial sections were cut 1.5 ~x thick and sta,ined with the pexiodie-Schiff reaction for insoluble carbohydrates, followed by Aniline blue black for proteins (FIs~E~, 1969). Results A u t o r a d i o g r a p h y of D N A - b o u n d 3I:[-actinomycin D in sections of freeze-substituted nucelli resulted in preparations similar to t h a t shown

124

D.B. FIsH~ grid W. A. JE~sn~:

in Fig. 1. Nuclei of the nucellar cells are heavily labelled and even the nuclei of the egg apparatus, in which DNA is notoriously difficult to demonstrate (J]s~s]~N, 1965b), showed obvious concentrations of silver gra~ins. With a few exceptions, the degenerated synergid contained two strongly labelled bodies in charaetexistic positions. I n one case three

Fig. 1 a and b. Antor~diograph of ~ 3 ~ paraffin sec'~ionfrom a freeze-substituted egg apparatus which had been incubated after sectioning with 3H-actinomycin D. X x-body, Z zygote nucleus, PN polar nuclei, a Unstained; b stained with Azure B. • 640 were present. One was almost always at the chalazal end of the synergid and the other about halfway up, near the surface of the synergid toward the central cell. Fig. 2 gives a diagrammatic s u m m a r y of the positions of these bodies in nine degenei~ated synergids. When these preparations were stained with Azure B, the '~H-actinonwcin D-labelled structures usually showed patches of the green color characteristic of the presence of DNA although the silver grains sometimes prevented its detection. Treatment of sections with DNase prior to incubation in 3tt-aetinomycin D eliminated the labelling of most nucellar cell nuclei. However, the two structures in the s~mergid retained their c a p a c g y to bind 3H-actinomycin D as did the degenerated nuclei in nucellar cells next to the

Cotton Embryogenesis: The X-Bodies

125

pollen tube and in the damaged outer cell layer of the nucellus. This is not unexpected as the DNA in pycnotie nuclei is usually resistant to DNase. Surprisingly enough, however, the DNA of the polar nuclei, synergid nucleus and the egg nucleus itself was also apparently resistant to DNase since these nuclei still showed obvious concentrations of silver grains. T h a t the labelling was due to DNA was demonstrated by extraction of sections with hot perehlorie aeid, which eliminated the capacity of all structures to bind DNA. DNA was also localized by the Feulgen reaction which resulted in intense staining of the structures identified as x-bodies in similar sections

Fig. 2. Diagramatie summary of the positions of 3H-actinomyein D-binding structures in 9 degenerated synergids. The zygote with its nucleus is at the left stained with PAS-Aniline blue black. This positive reaction is somewhat ambiguous, however, since the entire degenerated synergid stains moderately without prior acid hydrolysis, with the x-bodies staining more intensely than other inclusions. Their reactivity is considerably enhanced by acid hydrolysis. There can be no doubt, then, of the characteristic presence in the degenerated synergid of two DNA-eontMning bodies which are in fact identical to structures identified earlier as "x-bodies". The most likely candidates for their identity are the synergid nucleus and the vegetative nucleus from the discharged pollen tube. That this is the ease could be determined from a characteristic morphological difference between the two, due to the presence of a large nucleolus in the synergid nucleus (Fig. 3b) and the complete absence of a nueleolus in the vegetative nucleus (Fig. 3b, Jg~SEN and F I S ~ R , 1969). On this basis it could be shown t h a t the lower (micropylar) x-body (Fig. 3a) has an elhpticM body within it which did not bind Actinomyein D, stained only lightly after Feulgen hydrolysis and stained blue with Azure B. The remainder of the x-body is irregular in outline and did not stain uniformly. The former structure is lacking in the ehMazM x-body, which is irregular

126

D . B . FmH~R and W. A. JE~WSE~:

Figs. 3 a, b and 4 ~, b

Cotton Embryogenesis: The X-Bodies

I27

in outline a n d stains in p a t c h e s t h r o u g h o u t (Fig. 4a). This distinction b e t w e e n t h e x-bodies could be perceived in all b u t a few instances. T h a t t h e p y e n o t i c v e g e t a t i v e a n d synergid nuclei o c c u p y characteristic positions in t h e d e g e n e r a t e d synergid after p o l l e n - t u b e discharge can r e a d i l y be seen from Fig. 5. The d a t a used to c o n s t r u c t this d i a g r a m were t a k e n from 19 e m b r y o s a e s which h a d been either g l u t a r a l d e h y d e fixed or f r e e z e - s u b s t i t u t e d a n d e m b e d d e d in E p o n . The s y n e r g i d nucleus t y p i c a l l y lies in t h e central half of t h e s y n e r g i d on t h e side t o w a r d t h e c e n t r a l cell while t h e v e g e t a t i v e nucleus lies in t h e synergid tip. B o t h nuclei lie a t t h e edge of t h e zone occupied b y t h e P A S - p o s i t i v e m a t e r i a l e j e c t e d f r o m the pollen t u b e (Figs. 3a, 4a, JE~SEN a n d FISREI~, 1968a),

OID

O

Fig. 5. Diagrammatic summary of the positions of the degenerated vegetative nuclei (darkened circles) and synergid nuclei (open circles) in 19 degenerated synergids. The zygote with its nucleus is at the left or are p a r t l y s u r r o u n d e d b y p o l l e n - t u b e discharge. I n t h e single case where t h r e e s t r u c t u r e s in a s y n e r g i d were f o u n d to b i n d 3 t t - a c t i n o m y e i n D, two of t h e m were w i t h i n t h e p o l l e n - t u b e discharge, p e r h a p s i n d i c a t i n g some u n u s u a l e v e n t occurred, causing f r a g m e n t a t i o n of one of t h e nuclei. The use of f r e e z e - s u b s t i t u t e d a n d E p o n - e m b e d d e d tissue r e s u l t e d in two a d d i t i o n a l o b s e r v a t i o n s n o t m a d e w i t h g l u t a r a l d e h y d e - f i x e d ,

Fig. 3a and b. Comparison of the lower (micropylar) x-body (double arrow, 3a) with a synergid nucleus (double arrow, 3b). Most of the degenerated synergid is occupied by materials discharged from the pollen tube, and appears as the light-anddark-mottled area. Starch grains from the synergid are indicated by single arrows. Part of the pollen tube is visible of the lower right of the synergid. Aniline blue black-stained 1.5-tz Epon sections of glutaraldehyde-fixed material. • 1200 Fig. 4a and b. Comparison of the upper (ehalazM) x-body (double arrow, 4a) with a vegetative nucleus in the pollen tube (double arrow, 4b). This section comes from the same synergid as that shown in Fig. 3a. Starch grains from the synergid are indicated by single arrows. Part of the pollen tube is visible at the lower right of the synergid (a). The two sperm are visible in b (arrows). Aniline blueblack-stained 1.5 ~xEpon sections of glutaraldehyde-fixed material. X 1200

128

D.B. FISHERand W. A. JENSEN:

Epon-embedded material. The first of these concerns the state of the synergids before pollen-tube discharge. I n contrast to our earlier finding t h a t one of the synergids has always begun to degenerate before arrival of the pollen tube, freeze-substituted preparations were equa]ly consistent in showing no sign of degeneration in either synergid before discharge. This was true even in the cases in which the pollen tube had grown into the nucellus but had not yet penetrated a synergid. These observations were made with Aniline-blue-black-PAS-stained material with the light microscope. The second observation is on freeze-substituted, Epon-embedded tissue which contains appreciable amounts of an intensely PAS-positive material (Fig. 7) in the vacuoles of the synergids. These substances had not previously been observed in any of our preparations, including those which had been freeze-substituted and embedded in paraffin rather than Epon. This is almost certainly a water-soluble polysaccharide which is retained under the anhydrous conditions of the procedure (RoELOFSEN, 1959) and remains in the section because of physical restraints imposed by the epoxy resin. Several similar examples of PAS-positive material have been found in this laboratory in the course of using freeze-substituted, Epon-embedded tissue. The present work indicates that when the pollen tube discharges, some of the material of the degenerate synergid is apparently forced from the synergid (Fig. 6). This material does not enter any of the ceils of the embryosac, but passes over the top of the persistent synergid in the angle formed between the egg, the central cell and the persistent synergid (compare Figs. 6 and 7). This undoubtedly represents the path of least resistance for any material leaving the synergid tip. Since it stains fairly uniformly in electron micrographs, with only an occasional inclusion present, including a few vesicles (Fig. 8), it does not appear to originate from the pollen tube and is more probably some of the contents of the chalazal synergid. However, occasional polysaccharide granules, definitely from the pollen tube (JE~s]sN and FISH]~, 1968a), are present.

Fig. 6. A 1.5 [z Epon section through a freeze-substituted egg apparatus, showing the darkly-stained material (double arrow) which extends from the tip of the degenerated synergid (D.Syn) over the top of the persistent synergid (P. Syn) and down its side. Stained with PAS and Aniline blue black. • 680 Fig. 7. A freeze-substituted egg apparatus sectioned at right angles to the one shown in Fig. 6. The darkly stained material of Fig. 6 (double arrows) appears in the angle between the cytoplasms of zygote (Z), persistent synergid (P. Syn) and central cell. Intensely PAS-positive material in the synergid vacuoles, apparently a water-soluble polysaeeharide, is found in the persistent synergid (P. Syn). Stained with PAS and Aniline blue black. • 680

Cotton Embryogenesis: The X-Bodies

Figs. 6 and 7 9 Planta (Berl.), Bd. 84

129

Fig. 8. Electron micrograph of the darkly staining material forced from the synergid tip by the discharge of the pollen tube. A few polysaecharide vesicles (arrows) from the pollen tube are present. Part of the cytoplasm of the persistent synergid (P.Syn) and of the (Egg) and ~ male nucleus (Nu) are included. Glutaratdehydeosmium fixation. • 9260

D. B. FISHERand W. A. JENSEN: Cottort Embryogenesis: The X-Bodies

131

Discussion

In spite of the staining peculiarities of the degenerated synergid after pollen tube discharge, there is no question of the presence of DNA in the two structures previously identified as "x-bodies" (JE~s~N and FISKW~, 1968a). The results with the three stains employed for investigating this question were in complete agreement, in spite of the widely different staining mechanisms involved. Azure B acts as a basic dye (see JESSe?C, 1962), the Feulgen reaction relies on aldehyde groups produced as a result of depurinization of DNA by acid treatment (see J ] ~ S~N, 1962) and actinomycin D binds to the base guanosine in DNA (R~,Ic~ and GOLDBEI~G, 1964).] What, then, is the fate of the cytoplasmic sheaths surrounding the sperm ? Until they o r their organelles can be seen after pollen tube discharge, their fate must remain uncertain. Although we have never seen any sign of sperm cytoplasm in either the egg or central cell cytoplasms, both are large ceils and the few identifiable organelles from the male cytoplasm (JE~sEN and FISHnR, 1968c) might be overlooked. The situation is equally difficult if the sheaths remain in the degenerated synergid since they are small and not at all dense, and considerable shrinkage and degeneration is to be expected. It is possible that the two cytoplasmic structures we reported earlier (JE~s]~N and FIsHE~, 1968a) were the cytoplasmic sheaths of the sperm. There are several indications that the sperm move to the synergid tip before fusing with the egg and central cell. Perhaps the most convincing evidence for this movement of the sperm comes from the demonstration that the vegetative nucleus is consistently found in the tip after discharge. During the growth of the pollen tube the vegetative nucleus and sperm are always in intimate association in the pollen tube (Fig. 4b, JE~SE~ and FISHE~, 1968b) and one would expect them to move to similar locations in the synergid when the pollen tube discharged. Movements of the sperm to such a restricted space in the synergid tip would obviously enhance the chances of their contacting both the egg and the central cell. The identification of the synergid and vegetative nuclei in the degenerated synergid allows a tentative reconstruction of some events which occur during pollen-tube discharge. The synergid plastids with their starch grains provide another marker since they are found mostly in the vicinity of the filiform apparatus ( J ~ s E N , 1965a). As the pollen tube grows through the transmitting tissue of the style, the vegetative nucleus and sperm are several hundred microns behind the tip and the tube cytoplasm contains considerable amounts of PAS-positive materials ( J ~ S E ~ and FISHER, 1969). This situation is probably maintained until 9*

132

D.B. FISHERand W. A. JEssE,:

discharge and it appears likely that the vegetative nucleus and sperm are an appreciable distance from the tip, with a considerable volume of cytoplasm between. If the contents are discharged in order of their distance from the tip, all of the cytoplasm must be accommodated by the synergid before the sperm and the vegetative nucleus even leave the pollen tube. In spite of this the synergid actually decreases in volume (J~NsE~ and FrSHER, 1968a) and the vegetative nucleus, and most likely the sperm as well, make their way to the synergid tip. The present observations indicate that the materials ejected from the pollen tube push the synergid contents aside rather than ahead of the emerging pollen tube cytoplasm. Presumably, the first pollen tube contents follow in this lateral movement and cytoplasm ejected later moves further into the synergid. The result would be a fountain-like flow in which the materials farthest from the pollen-tube tip actually penetrate closest to the synergid tip. This might occur if the pollen-tube and synergid cytoplasms tended to gel on contact, with the result that only the pollen cytoplasm which had not yet contacted the synergid contents retained its capacity to flow. That the degenerated synergid is in a gel-like condition after discharge is indicated by the fact that no changes can be observed in it for several days after fertilization (JEsss~ and FrSHE~, 1968a). A possible indication of at least one kind of interaction between the pollen tube and synergid may be indicated by the difference between glutaraldehyde-fixed and freeze-substituted synergids. In the latter case, neither synergid shows any evidence of change before the pollen tube arrives, while in the glutaraldehyde-fixed material one of the synergids always shows varying degrees of degeneration before arrival of the pollen tube ( J ~ s ~ N and FIS~E~, 1968a). These changes seem to be intimately associated with the behavior of the pollen tube during its penetration of the egg apparatus. Since observations in both cases were consistent, it appears that glutaraldehyde fixation may cause some subtle change, which has already taken place in one of the synergids, to be visualized, perhaps by mimicking some effects of the pollen tube. I t is an interesting coincidence that the outer layer of the pollen-tube wall in the transmitting tissue (J]~SE~, FISJ~E~ and ASHTO~r unpublished) and in the nucellus contains aldehyde groups, as shown by its dimedonesensitive staining with Schiff reagent. Since this wall extends all the way to the tip, the presumably aldehydic materials entering into its construction would be the first to be ejected into the synergid. This work was supported in part by Public Health Service fellowship 5-F2-GM22,031-02 to D. B. FISKnRfrom the National Institute of General Medical Sciences, and in part by NSF Grant GB 6268 to W. A. JE~SECr

Cotton Embryogenesis: The X-Bodies

133

Rele~'ences CA~[ARGO, E. P., and W. PLANT: The radioautographie detection of DNA with tritiated actinomycin D. J. Cell Biol. 35, 713--716 (1967). CA~o, L. G , and 1%.P. yon TUBERGEI'~: High resolution autoradiography. I. Methods. J. Cell Biol. 16, 173--188 (1962). EBSTEtN, B. S. : Tritiated Actinomycin D as a eyt,ochemical label for small amounts of DNA. J. Celt Biol. 36, 709--713 (1967). FIS]ZER., D. B. : Protein staining of ribboned epon sections for light microscopy. Histochemie, in press (1969). J E N S ~*, W. A. : Botanical Histoehemistry. San Francisco: Freeman 1962. - - The ultrastructure and histochemistry of the synergids of cotton. Amer. J. Bot. 52, 238~-256 (1965a). - - The ultrastructure and composition of the egg and central cell of cotton. Amer. J. Bot, 52, 781--797 (1965b). - - and D. B. FISt~E~: Cotton embryogenesis: The entrance and discharge of the pollen tube in the embryo sac. Planta (Berh) 78, 158--183 (1968a). -Cotton embryogenesis: The sperm. Protoplasma 66, 277--286 (I968b). --Cotton embryogencsis: Double fertilization. Phytomorpbology, in press (1968e). - - - - Cotton embryogenesis: The pollen tube in the stigma and style. Protoplasma, in press (1969). MAttESHWARI, P. : An Introduction to the Embryology of the Angiosperms. New York: McGraw-Hill 1950. REICh, E., and I. H. GOLDBERG: Actinomycin and nucleic acid function. Progr. Nucleic Acid Res. and MoI. Biol. 3, 183--234 (1964). I~OELOFSEI%P. A.: The Plant Cell Wall. In: Encyclopedia of Plant Anatomy (W. ZI~ERMANN and P. G. OZEND.~, eds.), voh 3, pt. 4. Berlin: Borntraeger 1959. Prof. WmLIA~ A. JENSEN Department of Botany University of California Berkeley, California 94720, USA

Dr. DONALD B. FISTIER Department of Botany University of Georgia Athens, Georgia 30691, USA

Cotton embryogenesis: The identification, as nuclei, of the X-bodies in the degenerated synergid.

The "x-bodies" present in the degenerated synergid of cotton were shown to contain DNA by specific staining with Azure B, the Feulgen procedure, and b...
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