Arch. Histol. Cytol., Vol. 55, Suppl. (1992) p.

217-224

Ultrastructural in Angiosperms

Organization

of Two

Tapetal

Types

Susan

H.

BARNES

and

Stephen

BLACKMORE

The Natural

History Museum, London, United Kingdom

Received February 18, 1992

Summary. that include for studying scanning

The

development

of preparation

techniques

freeze fracturing the differentiation microscope.

provide an ideal method of plant tissues in the This is illustrated with

electron

reference to tapetal development in Catananche caerulea, which has a plasmodial tapetum, and in Lolium perenne, which has a secretory tapetum.

The tapetum is a specialised tissue concerned with the nutrition of the developing spores, and is found in the sporangia of lower plants and anthers of higher plants (for reviews, see PACINI et al., 1985; CHAPMAN, 1987). Tapetal cells exhibit a variety of developmental pathways, especially in terms of nuclear divisions, the behaviour of the cell walls and the synthesis of pollen wall precursors. Two major ultrastructural classes of tapetum are found in the anthers of angiosperms. The secretory tapetum is characterised by persistent cell walls and is also referred to as glandular, parietal, or cellular. In contrast, the cells walls of the plasmodia) tapetum break down during microspore development. This second kind of tapetum is sometimes referred to as invasive or amoeboid. The two major classes can themselves be subdivided on the basis of various criteria, particularly the sequence of events during microsporogenesis (PACINI et al., 1985; PACINI, 1990; PACINI and FRANCHI (1991). In the course of a programme of comparative studies of pollen ontogeny aimed at determining the morphogenetic pathways associated with taxonspecific features, we have examined representatives of the two major tapetal classes. Here, we describe and illustrate the later, mainly post tetrad, developmental stages of the secretory tapetum of Lolium perenne L. (Gramineae) and the plasmodial tapetum of Catananche caerulea L. (Compositae : Lactuceae), as studied by means of scanning electron microscopy.
217

As has been the case for other tissues within the developing anther, previous studies have documented the organization of the tapetum at the level of optical microscopy (see for example, SCHNARF,1923; UBISCH, 1927) and through the application of transmission electron microscopy (see for example, DICKINSONand LEWIS, 1973, PACINI and KEIJZER, 1989; EL-GHAZALY and NILSSON, 1991). Scanning electron microscope studies had to await the development of techniques which permitted the examination of the internal surfaces of organs, tissues, cells and organelles. We have reviewed the historical development of such techniques in relation to plant ultrastructure (BARNES and BLACKMORE,1986a) and emphasised the importance of the pioneering work of Professor Keiichi TANAKA. He devised a series of techniques ranging from cracking resin embedded blocks (TANAKA, 1974) through to methods involving chemical fixation followed by freeze fracturing (TANAKA, 1981; TANAKA and NAGURO, 1981). Advances in specimen handling and processing have enabled the basic technique to be applied to free cells suspended in culture medium (FUKUDOMEand TANAKA, 1986). Although initially applied to animal tissues, the basic technique proved readily adaptable to plant tissues by adopting an extended period of extraction in osmium tetroxide (BARNES and BLACKMORE, 1984a, b). One area of botanical research where the technique, which we refer to as freeze-fracture and cytoplasmic maceration, has proved particularly suitable has been the study of pollen and spore ontogeny (BLACKMOREand BARNES, 1985, 1987, 1990; BARNES and BLACKMORE,1986b; DICKINSON and SHELDON, 1986; WILMS et al., 1986). As observations of the tapetum demonstrate, the freeze fracture and cytoplasmic maceration technique is particularly suited to the study of the spatial relations of membrane-bound organelles.

218

S. H. BARNES and S. BLACKMORE:

MATERIALS

AND

METHODS

Anthers at various stages of development were taken from plants of Lolium perenne L. (Gramineae) collected from wild populations in Sussex and from specimens of Catananche caerulea L. (Compositae: Lactuceae) cultivated at Chelsea Physic Gardens, London. The anthers were prepared by the freeze fracture and cytoplasmic maceration technique as described by BARNES and BLACKMORE (1984a). Catananche anthers at later stages of development were dissected from the bud and individually trimmed at each end to assist the penetration of solutions. Early stage anthers were processed in buds or groups of anthers to facilitate easy handling. Lolium anthers were dissected and processed singly. Prepared anthers were fixed in 1% osmium tetroxide in M/15 phosphate buffer for 2-16 h with continuous rotation. They were then washed in buffer and transferred through 15%, 30% and 50% dimethyl sulphoxide for 30 min in each solution. The anthers were freezefractured on a liquid nitrogen-cooled metal block using a razor blade and hammer. The fragments were collected and thawed in fresh 50% dimethyl sulphoxide. After copious washing in buffer the specimens were transferred to 0.1% osmium tetroxide in M/15 phosphate buffer and left to macerate at 4C for 14 days. During this period the specimens were regularly checked and the solution replenished if it had discoloured. To enhance electrical conductivity the specimens were fixed in 1 % osmium tetroxide, washed, treated with 2% tannic acid, washed again and refixed in 1% osmium tetroxide. They were then dehydrated by transfer through an acetone series and critical point dried. The pieces were mounted on to specimen stubs using Araldite adhesive and sputter coated with approximately 15 nm of gold/palladium. Images were taken using a Hitachi 5800 field emission scanning electron microscope at an accelerating voltage of 8 kV.

RESULTS

AND

DISCUSSION

Plasmodial

tapetum

When the anther tissues first differentiate, the tapetal cells form a cylinder surrounded by both the parietal cells and the epidermal cells and enclosing the microsporocytes. This configuration (Figs. 1, 2) initially occurs in both major classes of tapetum. In the Compositae, and in 31 other flowering plant families with

plasmodial tapeta (ALBERTINIet al., 1987), the tapetal cells subsequently intrude into the spaces between developing microspores. In contrast, the tapetum remains an organized cylindrical tissue in the anthers of flowering plants with secretory tapeta, recognized from 175 families (ALBERTINI et al., 1987). In Catananche, as in Cichorium (PAcINI and KEIJZER, 1989), the tapetum does not become invasive until after the tetrad stage (Fig. 2). During the tetrad stage the tapetal cell walls remain intact and distinct. The nuclei of most, if not all, tapetal cells undergo mitosis during this stage. As in other tapetal cells (see for example, CHAPMAN, 1987), the nuclear division is not followed by cytokinesis. The cells are therefore binucleate, with the two nuclei remaining in close contact. After the callose special cell wall around the tetrads disperses, the free microspores are released into the locale (Fig. 3). The developing microspores have a spiny exine by this stage, the outer layer of the exine (the ectexine) is not fully differentiated (Fig. 4). The tapetum starts to intrude between the microspores early in the free microspore stage. Each tapetal cell has a continuous plasma membrane and has an organelle-rich cytoplasm, with particularly abundant endoplasmic reticulum (Fig. 5). At this stage of development the tapetum is generally considered to be a highly active secretory tissue producing sporopollenin precursors which form the exine. Consistent with this interpretation, numerous dictyosomes are present in the tapetal cytoplasm (Fig. 6). As the microspores develop (Figs. 7, 8) the exine differentiates and becomes caveate and the microspore nuclei enlarge. At this stage, the tapetum intrudes between the microspores but retains a continuous plasma membrane around its convoluted inner surface. As the tapetal cells extend towards the centre of the anther locule their large nuclei maintain a peripheral position. The tapetal cytoplasm (Fig. 8) continues to be actively synthetic and retains a highly organized system of endoplasmic reticulum. Once the exine reaches its mature morphology, the microspores enter a characteristic vacuolate stage (Fig. 9) in which the microspore cytoplasm is displaced to the periphery by a large vacuole. The tapetum, having completed its contribution to exine synthesis, begins to degenerate. The most conspicuous change, at this stage, is the absence of extensive sheets of endoplasmic reticulum (Fig. 10). Numerous vesicles are now present in the tapetal cytoplasm and the plasma membranes are no longer continuous. The freeze fracture and cytoplasmic maceration technique reveals fine details of the pollen wall, such as the minute cavities known as internal foraminae (SKVARLAand TURNER, 1966) present within elements

Two

Tapetal

Types

in Angiosperms

219

Figs.

1-6.

Catananche

caerulea.

Fig.

1. Premeiotic

anther

locule.

x 960. Fig.

2. Locule

at tetrad

stage,

tapetum of

binucleate. microspore Abbreviations: cyte,

x 880. Fig. 3. Locule with free microspores. x 880. Fig. 4. Detail of Fig. and tapetum from Fig. 3. x 8,800. Fig. 6. Detail of tapetal dictyosomes A aperture, 0 orbicule, E exine, L lipid, ER endoplasmic reticulum, V vacuole. D dictyosome,

3. x 2,400. Fig. 5. Detail in Fig. 3. x 48,000 MS microsporo-

M microspore,

N nucleus,

T tapetum,

220

S. H. BARNES and S. BLACKMORE:

of the ectexine. At a later stage (Figs. 11, 12), the nucleus of each microspore undergoes mitosis, giving rise to a generative and a vegetative cell. Technically this division marks the transition from microspore to pollen grain.

Catananche, in common with other members of the Lactuceae, has tricellular pollen grains at maturity. This situation results from a further mitotic division of the generative cell (Fig. 11), giving rise to the male germ unit (BARNES and BLACKMORE,1987). By this

Fig. 7-12. and tapetum pollen.

Catananche from Fig.

caerulea. 9. Locule

Fig. with

7. Locule vacuolate

with

plasmodial of tricellular

tapetum pollen

and free grains.

microspores. x 560. Fig.

x 800. 12. Surface

Fig. 8. Detail of exine of mature

of Fig. 7. x 3,200. Fig. x 5,600

microspores

and degenerating

tapetum.

x 640. Fig. 10. Detail

9. x 8,000.

Fig.

11. Locule

Two

Tapetal

Types

in Angiosperms

221

stage the tapetum has almost completely degenerated. Some discrete organelles, notably mitochondria, are still present but lipid droplets are the most abundant features at this stage (Fig. 12). This process of degeneration gives rise to a oily surface coating,

known as Catananche, number of in nature, each other

pollenkitt. Entomophilous plants, such as have pollenkitt which contains a large lipid droplets and is therefore very sticky thus allowing pollen grains to adhere to to aid dispersal by insects.

Figs. 13-18. Lolium perenne. Fig. aperture towards tapetal membrane. Epidermal membranes and tapetal cells. cytoplasm. x 5,600. in tapetal

x180. Fig. 13. Anther at vacuolate microspore stage. x 2,000. Fig. 15. Tapetal membrane and organelles. Fig. 17. Amyloplast in detail of Fig. 16. x 20,000. x 28,000

14. Orientation x12,000. Fig. Fig. 18. Concentric

of 16.

222

S. H. BARNES and S. BLACKMORE:

Secretory

tapetum

Development of the anther of Lolium perenne, has been investigated in detail, with special reference to the tapetum (PAcINI et al., 1992) and to pollen devel-

opment (VITHANAGE and KNOX, 1980). The SEM observations presented here represent a selection of the later developmental stages and supplement the detailed findings of the transmission electron microscope studies.

Figs. 19-24. 19. x 3,200. chloroplasts 23. x 16,000

Lolium Fig. 21. of parietal

perenne. Fig. Pollen grains cell. x 12,000.

19. Locule with almost mature pollen and degenerating tapetum. x 1,600. Fig. 23. Inner surface of tapetal

grains. x 480. Fig. 20. Detail of Fig. Fig. 22. Detail of Fig. 21. showing x 2,800. Fig. 24. Detail of Fig.

membrane

Two

Tapetal

Types

in Angiosperms

223

The anther of Lolium has four locules with a narrow central connective (Fig. 13). The anther wall is relatively thin, consisting of an outer epidermal layer, with a very narrow layer of parietal and tapetal cells. The centre of each locule is a large space, which is filled with fluid until shortly before anther dehiscence. The developing microspores exhibit a special orientation with respect to the tapetum, as in other Gramineae (CHRISTENSEN and HORNER, 1974). Each microspore lies with the single distal porate aperture in intimate contact with the tapetal membrane (Fig. 14). This arragement has been considered by PACINI (1990) and others to facilitate the uptake of nutrients which pass from the tapetum through the aperture into the microspore. This tapetal membrane is of considerable interest, because, in contrast to that of the plasmodial tapetum, it develops special accretions of sporopollenin. These structures, termed Ubisch bodies, were described by VON UBISCHin 1927. Ubisch bodies (Fig. 15) are characteristically found in seed plants with secretory tapeta (PACINI, 1990) and similar structures occur in pteridophytes with secretory tapeta (LUGARDON, 1981). The organelles within the cytoplasm of the secretory tapetum differ from those of the plasmodial tapetum. Rather than the abundant sheets of rough endoplasmic reticulam, numerous plastids can be seen (Figs. 15-18). As PACINI et al. (1992) have documented in Lolium, there is a transition from chromoplasts to elaioplasts during microspore development. In the shallow, spreading cells of the tapetum (Fig. 16) the disposition of the organelles and their products can be seen. These include numerous amyloplasts (Fig. 17) and abundant lipid droplets. Such tapetal accumulations of lipid are temporary, and are normally considered to be absorbed by the developing pollen grains (PACINI, 1990). Thus, when degeneration of the taptum takes place, these lipids are absorbed into the pollen grains, rather than contributing to a surface pollenkitt. The tapetal cytoplasm also contains tightly packed concentric membranes that may themselves be derived from plastids (Fig. 18). In anthers with almost mature pollen prams (Figs. 19-24) further details of the relationship between the tapetum and pollen can be observed. As in plasmodial tapeta, the nuclei of the tapetum undergo incomplete mitotic division (Figs. 19, 20) after which the chromosomes remain partially condensed (Fig. 21). The contrasting ultrastructure of parietal cells and tapetal cells illustrates the differing developmental programme of each tissue. Parietal cells (Figs. 21, 22) contain functional chloroplasts of typical form. The degeneration of the tapetum has progressed to the

point where large lipidic droplets are the most conspicuous feature. Lolium like other members of the Gramineae is anemophilous. The products of tapetal degeneration are absorbed by the pollen grains leaving no tapetal remnant except the Ubisch bodies (HESSE, 1980; PACINI, 1990), consequently the pollen grains are devoid of sticky pollenkitt and are readily dispersed by the wind. The inner surface of the tapetal membrane after anther dehiscence (Figs. 23, 24) shows clearly, the boundaries between individual tapetal cells. No special features are recognizable that would indicate the points of contact of the pollen apertures. At higher magnification it can be seen that the orbicules are attached to a sporopollenin membrane with a somewhat reticulate organization.

CONCLUSIONS The example of the tapetum demonstrates the value of the freeze fracture and cytoplasmic maceration technique as a method of studying the developmental programmes of various as the anther.
Acknowledgements.

tissues

within

an organ

such

We thank

Prof.

Keiichi

TANAKA for Museum to pollen Garden

his advice during a visit to the Natural History in 1983 which inspired us to apply his techniques development. We are grateful to Chelsea for the provision of plant material. Physic

REFERENCES

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224

S. H. BARNES and S. BLACKMORE

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Dr. Susan H. BARNES The Natural History Museum Cromwell Road London SW7 5BD United Kingdom