m. r. vijayaraghavan and tripat kapoor

Cytologia 50: 333-340, 1985

Nucleocytoplasmic Interaction in the Embryo-basal Cell

and in the Chalazal-endosperm Chamber Cell

in Potamogeton nodosus

M. R. Vijayaraghavan and Tripat Kapoor

Department of Botany, University of Delhi, Delhi 110007, India

Accepted November 29, 1983

After fertilization, the embryo and endosperm develop. The angiosperm embryogenesis culminates in the differentiation of embryo into two parts: namely the embryo-proper and the embryo-suspensor. The endosperm develops either by nuclear, cellular or helobial ontogeny. In Potamogeton nodosus, the endosperm development follows the helobial type and the chalazal endosperm chamber/cell remains undivided (Fig. 1). Classical studies assign the suspensor cell/cells the role of pushing the embryo deep into the endosperm and the endosperm plays an active role in nourishing the embryo. During the study of embryology and cytochemistry of Potamogeton nodosus, it was seen that both in embryo-basal cell and the chalazal endosperm chamber/cell, homogeneous material extruded from the nucleolus through nucleoplasm into the cytoplasm.

Nucleolar extrusions into the cytoplasm are known both in plants and animals. The nature of extruded substances are variously interpreted: as cytoplasmic-radiations (Bambacioni and Giombini 1930), cytoplasmic-globules (Sargent 1896) and thick, perinuclear layers of cytoplasm (Cooper 1935). The role of these extruded materials is also subjected to speculation (see Gates 1942). Little cytochemical and ultrastructural work has been done to determine the nature of these substances. This present communication deals with the cytochemical nature of such nucleolar extrusions, in the embryo-basal cell and chalazal endosperm chamber.

Material and methods

Fruits, at various stages of development, of Potamogeton nodosus Poir were

fixed in FAA (Formalin-acetic-alcohol), 10% neutral formlin and AA (Acetic acid

alcohol) and dehydrated in tertiary butyl-alcohol series and embedded in paraffin

wax. The sections were cut at 10ƒÊ thick. Materials fixed in FAA and in neutral

formalin were used for the localization of proteins with mercuric-bromophenol blue

method (Mazia et al. 1953), RNA by pyronin-Y method (Tepper and Gifford

1962) and DNA by Feulgen reaction (Kallarackal 1974). Control slides were

made to check the specificity of the various reactions.

Observations

Basal cell of the embryo

The zygote divides to form the basal cell and the terminal cell (Fig. 2A). The

334 M. R. Vijayaraghavan and Tripat Kapoor Cytologia 50

Fig. 1. The embryo sac of Potamogeton nodosus showing globular proembryo

with basal cell, micropylar and chalazal endosperm chamber (diagrammatic presentation). (bc, basal cell; bn, basal cell

nucleus; cc, chalazal endosperm chamber; cn, nucleus of chalazal endosperm chamber; mc, micropylar endosperm chamber; mn, nucleus of micropylar

endosperm chamber; nb, nucleolar bleb;

pe, globular proembryo).

basal cell never divides, but enlarges, de

velops into a conspicuous vesicular struc

ture (Figs. 1, 2B, D) and its nucleus is large,

polyploid having dense nucleoplasm (Fig.

2B).

At about globular proembryo stage, in

the basal cell, the nucleolus recrudesces and

blebs off many extrusions that migrate

towards the nuclear periphery (Fig. 2B).

These bodies stain densely for proteins (Fig.

2B), RNA and DNA (Fig. 2G, H). During

further embryogenesis, when the basal cell

as well as its nucleus enlarges the latter

shows irregular contour (Fig. 2C, E). The

nucleolus is in perpetual process of blebing

and the extruded substance is, however,

mostly homogeneous in nature. At the

monocotyledonous embryo stage, the nu

cleus is deeply lobed and the extrusions are

located at the lobed sites of the nucleus

which remain inside the nuclear periphery

(Fig. 2E, E•L). Thus, in a given nucleus,

the nucleolus shows many sites of blebing

(Fig. 2E, E•L, 1). The substance thus given

off from the nucleolus has a strong affinity

for proteins (Fig. 2C), RNA (Fig. E) and

Feulgen (Fig. 21) stainings.

At the embryonic shoot-apex stage of

embryo, these nucleolar extruded bodies are

seen in the cytoplasm (Fig. 2F) and also

stain for proteins, RNA (Fig. 2F) and DNA

(Fig. 2 J). The striking similarity between

the cytochemical staining of the nucleolar

Fig. 2 A-J. Nucleocytoplasmic interaction in basal cell of Potamogeton nodosus. A, two-celled

proembryo. The intensity of cytoplasmic and nucleolar RNA (pyronin-Y method) is denser in the

basal cell than in the terminal cell. •~880. B, basal cell at globular proembryo stage. Note

the distribution of nucleolar blebs at the periphery of the nucleus (MBB-test). •~400. C, basal

cell at monocot embryo stage to reveal the lobed nucleus and protein-rich (MBB-test) nucleolar

bleb (arrow) •~ 380. D, basal cell at monocot embryo stage when shoot-apex is well-differentiated.

MBB-positive nucleolar blebs (arrows) in the cytoplasm are noteworthy. •~40). E, basal cell at

monocot embryo stage. The nuclei (E, E•L) are deeply lobed, nucleolar blebs (arrows) are peri

pherally distributed and stain for RNA. •~350. F, basal cell at embryonic shoot-apex stage.

The RNA-stained nucleolar blebs are pinched off and these bodies migrate into the cytoplasm.

•~ 400. G, H, portions of basal cells at globular embryo stage to show Feulgen-positive nucleolar

blebs (arrows) at the periphery of the nucleus. •~400. I, J, portions of basal cells at later stages

of embryogenesis to show the Feulgen-positive nucleolar blebs (arrows) extruded from the nucleolus

(I) into the cytoplasm (J). •~400. (bc, basal cell; tc, terminal cell)

1985 Nucleocytoplasmic Interaction in Potamogeton nodosus 335


extrusions and the nucleolar-like bodies observed in the cytoplasm led us to consider

that the nucleolar-like bodies in the cytoplasm are pro parte nucleolar extrusions.

Thus nucleo-cytoplasmic interaction is established in the basal cell of Potamogeton

nodosus.

Nuclear wall lysis has not been observed for the migration of nucleolar ex

trusions into the cytoplasm. They probably migrate towards the nuclear wall

(Fig. 2B, E) and at the contact site disintegration of nuclear wall results the extrusion

of these bodies into the cytoplasm.

Chalazal endosperm chamber/cell

The primary endosperm nucleus that lies in the chalazal part of the embryo

sac divides followed by oblique wall. This results in the formation of a larger

micropylar chamber and a small chalazal chamber. Free nuclear divisions occur

in the micropylar chamber (Figs. 1, 3A). The chalazal endosperm chamber is wedge

shaped (Fig. 1), its nucleus does not divide further and remains uninucleate. The

development of endosperm in this taxon is of the Helobial type.

At about globular embryo stage, many small nucleolar extrusions are observed

at the periphery of the nucleus (Fig. 3B). These bodies stain intensely for proteins

(Fig. 3B) and RNA. The heterochromatin organizing centres are seen with Feulgen

stain (Fig. 3E, F). During embryogenesis and at the time of the initiation of em

bryonic shoot-apex and later stages, the nucleolar blebs are seen in the cytoplasm

(Fig. 3C, C•L arrows, D). These nucleolar extrusions stain for proteins (Fig. 3D),

RNA (Fig. 3C•L) and DNA (Fig. 3G arrows). The identical cytochemical nature

of these bodies found in the nucleoplasm and the cytoplasm led us to consider these

bodies as pro parte of nucleolus, thereby establishing nucleocytoplasmic interaction

in the chalazal endosperm chamber (cell) of Potamogeton nodosus.

Discussion

Nucleolar extrusion

Intense metabolic activity of the embryo-basal cell is known in many plants

(Avanzi et al. 1972, Clutter et al. 1972, Corsi et al. 1973, Nagl 1976, Yeung 1980).

One of the evidences for such activity is attributed to the extrusion of nucleolar

Fig. 3 A-G. Nucleocytoplasmic interaction in the chalazal endosperm chamber of Potamogeton

nodosus. A, a portion of the longisection of ovule to show micropylar (partly) and chalazal endo

sperm chambers (MBB-stained). •~130. B, portion of the chalazal endosperm chamber at

globular proembryo stage to show protein-rich (MBB-test) nucleolar bodies (arrows). •~400.

C, portion of chalazal endosperm cell at embryonic shoot-apex initiation stage of the embryo.

•~ 400. C•L shows RNA-rich (pyronin-Y method) nucleolar blebs (arrows) in the cytoplasm of the

chalazal endosperm chamber. •~350. D, same, at later stage of embryogenesis, to show numerous

protein-positive (MBB-test) nucleolar bodies (arrows) in the cytoplasm. •~180. E, F, portions

of chalazal endosperm chambers at globular proembryo stage. Note the heterochromatin

(Feulgen-reaction) organizing centres especially in F. •~380. G, same at a later stage of em

bryogenesis to show Feulgen-positive nucleolar blebs (arrows). Both nucleolar bodies reveal

active budding. •~400. (cc, chalazal endosperm chamber; cn, nucleus of chalazal endosperm

chamber; mc, micropylar endosperm chamber; mn, nucleus of micropylar endosperm chamber)


material into the cytoplasm (Avanzi et al. 1970, Nagl 1973). The nucleolar extrusions that are observed in the cytoplasm of basal cell and chalazal endosperm chamber in Potamogeton nodosus are also reported in Noctiluca scientillans (Afzelius 1963).


Szollosi (1965) interpreted the nucleolar extrusons, in rat, as a means of transport mechanism of nuclear material into the cytoplasm. In oocytes of Anagasta kuhniella, nucleolar extruded granules are encircled by double membrane and are then transferred to cytoplasm (Cruickshank 1972). Hay (1968) described that dispersed nucleolar material passes through nuclear pores and reaggregate in the perinuclear cytoplasm.

The behaviour and staining reaction of nucleolar extruded material of the nucleus of the chalazal endosperm chamber and the embryo-basal cell are similar. The extrusion of nuclear material has also been reported in the chalazal portion of endosperm tissue in Phaseolus coccineus (Cionini and Cremonini 1970), Capsella bursa pastoris (Schulz and Jensen 1973) and Gossypium hirsutum (Schulz and Jensen 1977).

Cytochemistry of nucleolar extruded materialAccording to Nagi (1970), in Phaseolus sp., a portion of the nucleolar extru

sion contains proteins and some RNA. Another portion originating from the heterochromatin contains in addition to proteins and RNA, demonstrable amount of DNA. The DNA of such micronucleoli of suspensor cells of Phaseolus coccineus are due to extra replication of both ribosomal and non-ribosomal RNA (Avanzi et al. 1970, 1972). Present work on Potamogeton nodosus, also indicates that the nucleolar extrusions into the cytoplasm besides staining for proteins and RNA, are also Feulgen-positive and thus they are histochemically same in composition as the nucleolus.

Nucleo-cytoplasmic interaction

In several Lilium spp., the phenomenon of nucleo-cytoplasmic interaction sug

gests that the homogeneous material extruded from the nucleolus into nucleoplasm and then into the cytoplasm, control many nuclear activities. They are believed to hold nuclei in position and presumably cause the three chalazal spindles, in

megasporocyte, to fuse (Flint and Johansen 1958). In Pteridium, such nucleo-cyto

plasmic interaction is concerned with a preparation for sporophytic growth (Bell 1972, 1975).

The nuclei of the basal cell and chalazal endosperm chamber, in Potamogeton

nodosus, show conspicuous invaginations at the monocot embryo stage. These nuclear invaginations do contain material derived from the nucleolus. Nuclei of

irregular outline, as observed in the basal cell and chalazal endosperm chamber of the investigated taxon, are generally associated with secretory function (Opik 1965, Schnepf and Nagl 1970).

It is significantly interesting in Potamogeton nodosus that the two cells which reveal nucleo-cytoplasmic interaction, only after fertilization, have different origin and cytology. The embryo-basal cell is derived from the diploid zygote whereas

the chalazal endosperm chamber cell is the product of unequal division of primary

endosperm nucleus (triploid) resulting from Helobial ontogeny. These two cells, namely the embryo-basal cell and the chalazal endosperm chamber (cell), although different in ontogeny and development behave similarly in the nucleolar activity,


cytology and histochemistry .The phenomenon of nucleo-cytoplasmic interaction as reported in the two

cells i.e. the basal cell and the chalazal endosperm cell in Potamogeton nodosus

(present work) and the granular extruded nucleolar material perhaps represents a modus operandi for the supply of ribosomes, RNA and proteins needed to support the initial rapid cell expansion of basal cell and chalazal endosperm chamber . Moreover, such nucleolar extruded materials into the cytoplasm of these two cells derived from different and distinct ontogeny and ploidy may help in the stopage of cytokinesis of the basal cell and chalazal endosperm chamber in the investigated taxon.

Summary

The two cells, basal and chalazal endosperm chamber in Potamogeton nodosus, although have different ontogeny and ploidy, yet reveal identical metabolic activity and cytochemical behaviour during progressive stages of embryogenesis.

At the globular proembryo stage, in the basal cell as well as in the chalazal endosperm cell, numerous nucleolar bodies aggregate at the periphery of the nucleus. The nuclei of both the cells at the monocot embryo stage, become deeply-lobed and produce nucleolar extrusions. These nucleolar bodies during later embryogenesis migrate into the cytoplasm, stain for proteins, RNA and are also Feulgenpositive. The phenomenon of nucleo-cytoplasmic interaction is, thus revealed. Such a phenomenon may help in the stopage of cytokinesis in the basal cell and the chalazal endosperm chamber (cell) in Potamogeton nodosus.

Acknowledgement

One of us (T. K.) is grateful to the Planning Unit, University of Delhi, for financial assistance.

Literature cited

Afzelius, B. A. 1963. The nucleus of Noctiluca scintillans. Aspects of nucleocytoplasmic exchanges and the formation of nuclear membrane. J. Cell Biol. 19: 229-238.

Avanzi, S., Cionini, P. G. and D'Amato, F. 1970. Cytochemical and autoradiographic analyses on the embryo suspensor cells of Phaseolus coccineus. Caryologia 23: 605-638.

- , - and - 1972. Cytological localization of ribosomal cistrons in polytene chromosomes of Phaseolus coccineus. Chromosoma 39: 191-203.

Bambacioni, V. and Giombini, A. 1930. Sullo sviluppo del gametofito femminile in Tulipa gesneriana L. Ann. di Bot. 18: 373-386.

Bell, P. R. 1972. Nucleocytoplasmic interaction in the eggs of Pteridium aquilinum maturing in the presence of thiouracil. J. Cell Sci. 11: 739-755.

- 1975. Physical interactions of nucleus and cytoplasm in plant cells. Endeavour 34: 19-22.Cionini, P. G. and Cremonini, R. 1970. Aleuni aspetti citological dell' endosperma di Phaseolus

coccineus L. G. Bot. Ital. 104: 483-491.Clutter, M., Brady, T., Sussex, I. and Walbot, V. 1972. Rates of RNA and DNA synthesis in

embryos and suspensors of Phaseolus coccineus. Am. J. Bot. 59: 648-649.Cooper, D. C. 1935. Macrosporogenesis and development of the embryo sac of Lilium henryi.


Bot. Gaz. 97: 346-355.Corsi, G., Renzoni, G. C. and Viegi, L. 1973. A DNA cytophotometric investigation on the

suspensor of Eruca sativa Miller. Caryologia 26: 531-540.Cruickshank, W. J. 1972. The formation of 'accessory nuclei' and annulate lamellae in the oocytes

of the floor moth Anagasta kuhniella. Z. Zellforsch. 130: 181-192.Flint, F. F. and Johansen, D. A. 1958. Nucleocytoplasmic relationship in the Fritillaria type of

megagametogenesis. Am. J. Bot. 45: 464-473.Gates, R. R. 1942. Nucleoli and related nuclear structures. Bat. Rev. 8: 337-409.Hay, E. D. 1968. Structure and function of the nucleolus in developing cells. In: The Nucleus

(eds. A. J. Dalton and F. Haguenau. pp. 1-79. Academic Press, New York.Kallarackal, J. 1974. A modified Schiff reagent for use in Feulgen reaction. Curr. Sci. 43: 120-121 .Mazia, D., Brewer, P. A. and Alfert, M. 1953. The cytochemical staining measurement of protein

with mercuric bromophenol blue. Biol. Bull. 104: 57-67.Nagl, W. 1970. Temperature-dependent functional structures in the polytene chromosomes of

Phaseolus, with special reference to the nucleolus organizers. J. Cell Sci. 6: 87-107.- 1973. Origin and fate of micronucleoli in the giant cells of Phaseolus suspensor. Nucleus 16:

100-109.- 1976. Early embryogenesis in Tropaeolum majus L.: Ultrastructure of the embryo-suspensor .

Biochem. Physiol. Pflanz. 170: 253-260.Opik, H. 1965. The form of nuclei in the storage cells of the cotyledons of germinating seeds of

Phaseolus vulgaris L. Exp. Cell Res. 38: 517-522.Sargent, E. 1896. The formation of sexual nuclei in Lilium martagon. I. Oogenesis. Ann. Bot .

10: 445-477.Schnepf, E. and Nagl, W. 1970. Uber einige Strukturbesonderheiten der Suspensorzellen von

Phaseolus vulgaris. Protoplasma 69: 133-143.Schulz, Sister Patricia and Jensen, W. A. 1973. Capsella embryogenesis: The central cell. J.

Cell Sci. 12: 741-763.- and - 1977. Cotton embryogenesis: The early development of the free nuclear endosperm .

Am. J. Bot. 64: 384-394.Szollosi, D. 1965. Extrusion of nucleoli from pronuclei of the rat . J. Cell Biol. 25: 545-562.Tepper, H. B. and Gifford, E. M. Jr. 1962. Detection of ribonucleic acid with pyronin . Stain

Technol. 37: 52-53.Yeung, E. C. 1980. Embryogeny of Phaseolus. The role of the suspensor . Z. Pfl. Physiol. 96:

17-28.

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