FINE STRUCTURE STUDY OF POLLEN DEVELOPMENT IN HAEMANTHUS KATHERINAE BAKER

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1 J. Cell Sci. 8, (1971) 289 Printed in Great Britain FINE STRUCTURE STUDY OF POLLEN DEVELOPMENT IN HAEMANTHUS KATHERINAE BAKER I. FORMATION OF VEGETATIVE AND GENERATIVE CELLS JEAN M. SANGER AND W.T.JACKSON Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire O3755, U.S.A. SUMMARY When microspores of the African blood lily divide, they form pollen grains which consist of 2 cells of unequal size. This is accomplished when the microspore nucleus is displaced from the centre of the grain prior to division. The displacement is always towards the side of the grain opposite the furrow, and large vacuoles form in the cytoplasm between the furrow and the nucleus. During cell division the cell plate curves around one daughter nucleus and fuses with the pollen wall to enclose the generative cell. The cell-plate attachment always occurs with the wall that is opposite the furrow of the grain. Most of the microspore's organelles become incorporated in the larger vegetative cell, whereas the generative cell has few, if any, plastids and only a small number of other organelles. The wall around the generative cell is composed of finely fibrillar material enclosed within 2 unit membranes. The generative cell eventually becomes detached from the pollen wall, becomes spheroidal, and moves to a position near the centre of the pollen grain. At the same time, the large vacuoles disappear from the vegetative cell and the number of organelles increases substantially. INTRODUCTION Cell division in microspores of flowering plants is noteworthy for several reasons. The division is a polar one, resulting in daughter cells of unequal size. Furthermore, the smaller of the 2 cells initially attaches to the pollen wall and later lies entirely within the cytoplasm of the larger cell. Finally, these 2 cells differ greatly in their subsequent development and functions. Most of the published ultrastructural studies of microspore division and early pollen development were undertaken without the benefit of glutaraldehyde-osmium fixation (Bopp-Hassenkamp, i960; Diers, 1963; Larson, 1965; Sassen, 1964; Maruyama, Gay & Kaufmann, 1965). Two exceptions are the recent studies of Heslop- Harrison (1968) and Angold (1968). The present study using glutaraldehyde fixation was undertaken to determine how the microspore nucleus is displaced before the polar division, what ultrastructural differences exist between the 2 cells, how the generative cell is attached to the pollen wall and, finally, in what manner it becomes detached. CE L 8

2 290 J. M. Sanger and W. T. Jackson MATERIALS AND METHODS The cells examined in this study were obtained from plants of the African blood lily, Haemanthus katherinae Baker, grown in the Murdough Experimental Greenhouse at Dartmouth College. The colour and length of the anthers give an approximate indication of their developmental stage. Thus, anthers change from white to yellow to orange as they mature to a final length of about 5 mm. To determine the exact stage of development, however, the contents of the anther were stained with acetocarmine and examined with the light microscope. Anthers which were to be examined in the electron microscope were first cut into pieces 1-2 mm long One piece was stained with acetocarmine to determine the developmental stage and the other pieces were fixed for 1 h at room temperature in 15 % glutaraldehyde buffered at ph 70 in 005 M phosphate buffer. The pieces were washed in at least 4 changes of phosphate buffer over a 1-2 h period before being post-fixed in 1 % OsO 4 for 1 h. Dehydration was accomplished in 5-min steps in a graded series of ethanol followed by two 5-min changes in propylene oxide. Two different plastic mixtures were used for embedding: (a) 1:1 mixture of Epon 812 and nadic methyl anhydride (Luft, 1961) and (b) Epon 812, Araldite 506, and dodecylsuccinic anhydride (Mollenhauer, 1964). Sections were cut with a diamond knife on an LKB Ultrotome III and picked up on uncoated 200-mesh grids. They were stained for 2 h in a saturated uranyl acetate-water solution then post-stained in Reynolds's lead citrate (Reynolds, 1963) before being examined with the Zeiss EM 9 A electron microscope. OBSERVATIONS A microspore of H. katlierinae about to undergo mitosis is characterized by a sculptured wall and a large nucleus which has been displaced from the cell's centre towards one side of the grain (Fig. 1). The nucleus is always displaced in a particular direction; that is, to the side of the grain opposite the furrow. Vacuoles form between the nucleus and the furrow. Present also at this stage is a scattering of lipid droplets, /<m in diameter, as well as other typical cell organelles. Plastids have few lamellae and contain large starch inclusions. By the time chromosome condensation has begun, the organelles exhibit a polarity in their cytoplasmic distribution (Fig. 2). Most of the plastids, mitochondria, and lipid bodies are displaced toward the furrow side of the cell. Around the nucleus is a narrow zone devoid of the larger organelles and containing short, unaligned microtubules (Fig. 3). The plastids lack starch at this stage but sometimes contain small osmiophilic bodies (Fig. 2) and often appear to be dividing (Fig. 4). As mitosis continues, the majority of organelles remain associated with the chromatin mass that becomes incorporated into the vegetative cell. In Fig. 5 there are no plastids on the generative cell's side of the cell plate. In fact, a plastid was seen in only 2 of all the sections of generative cells examined. Two-celled pollen grains fixed shortly after completion of mitosis contain spherical nuclei (Fig. 6). Large vacuoles are present in both cells, and the wall of the generative cell is attached to the pollen wall at points opposite the furrow. The boundary between generative and vegetative cytoplasms consists of 2 cell membranes separated by a gap which varies in width from 45 to 120 nm. The 2 cell membranes are the same width (10 nm) as the cell membrane which borders the intine. The outer leaflets of each cell membrane (i.e. those bordering the gap, as opposed to the cytoplasm) appear more dense. This is also true for the cell membrane leaflet which

3 Fine structure of developing pollen 291 borders the intine of the pollen grain. The generative wall consists of afinelyfibrillar component sandwiched between the 2 unit membranes (Figs. 7, 8). At the stage when the large vacuoles have disappeared from both cells of the pollen grain (Fig. 9), the generative cell detaches from the pollen wall and becomes surrounded by vegetative cytoplasm. Before detachment occurs, membrane fragments can be seen between the intine and the plasma membrane (Fig. 8). These fragments are concentrated along the junction between the generative cell and the pollen wall. They are not seen in other areas of the wall at this stage, nor are they seen during any other stages of pollen development. Microtubules are positioned along both sides of the generative cell at its junctions with the pollen wall. The areas of contact of generative cell wall and pollen wall move closer together (Figs. 6, 7) until the generative cell finally is pinched off. At this time it is spherical and moves to the centre of the pollen grain, where it is surrounded by lipid droplets (Fig. 9). The vegetative cell at this stage has lipid droplets scattered throughout the cytoplasm in addition to those which surround the generative cell. The cytoplasm contains large numbers of mitochondria and plastids which were not evident before generative cell detachment. DISCUSSION Cell division of a microspore produces a 2-celled pollen grain which early in its development consists of a cell within a cell. These 2 cells are destined to play quite different roles: the generative cell, embedded in the cytoplasm of the vegetative cell, produces 2 sperm by a subsequent division; and the vegetative cell produces the pollen tube in which the generative cell divides and through which the sperm gain access to the egg. That 2 such cells should differ markedly from the time of their origin is not surprising. This is readily documented by light and electron microscopy. The distribution of organelles during division of the microspore produces a vegetative cell containing most of the microspore's cytoplasm and a generative cell containing very little cytoplasm. There is no evidence from this study or other published reports (Angold, 1968; Heslop-Harrison, 1968) that a particular ultrastructural event can be causally related to the displacement of the nucleus prior to division. No peripheral microtubules were ever seen demarcating the boundary of the future cell plate as has been reported for the polar cell division giving rise to wheat stomatal cells (Pickett-Heaps & Northcote, 1966). The cell plate curving around the generative nucleus is hemispherical. The cell wall, which forms subsequently, consists of unidentified finely fibrillar material between 2 unit membranes. In some micrographs the material of the generative cell wall has the same appearance as the intine with which it is initially contiguous and which has been reported to be composed of cellulose and polyuronides (Bailey, i960). The generative cell wall in H. katherinae does not have the same electron density as is seen in the callose walls of the generative cells of orchid pollen (Heslop-Harrison, 1968). The detachment or pinching off of the generative cell follows the same general pattern 19-2

4 292 J. M. Sanger and W. T. Jackson as reported for orchid (Heslop-Harrison, 1968) and bluebell pollen (Angold, 1968). In addition, microtubules are present in H. katherinae at the junction of the generative cell wall and pollen wall and appear to run parallel to these walls. Since the microtubules are present both within and outside the generative cell at the junction it is difficult to assign a role to them in the detachment of the cell. The origin of the small vesicles found within the junction between the generative cell and pollen wall could not be determined. There was no indication that they derived from dictyosomes. Since the vesicles are present only prior to generative cell detachment, they may be contributing cell wall material to the generative cell. Heslop- Harrison (1968) has suggested that detachment of the orchid generative cell is caused by the growth of the generative cell wall over the surface of the intine, followed by the separation of the 2 walls. It is possible that such a process also occurs in H. katherinae, although the mechanism by which the generative wall changes from a hemispherical to a spherical state before detachment still remains to be explained. Plastids are excluded almost entirely from the generative cell. The localization of these organelles can be seen clearly in the microspore by telophase of the mitotic division. Some of the other types of cell organelles are included in the cytoplasm of the future generative cell during telophase and it is not clear how the plastids are excluded. They are much larger than any of the other cytoplasmic organelles and this may be a factor in their exclusion. The absence of plastids from generative cells has been reported in several other species of pollen (Sassen, 1964; Larson, 1965; Heslop-Harrison, 1968). During and for a time after microspore mitosis, plastids increase in number, apparently by division, and decrease in size. Starch is not present in the plastids of the newly formed pollen grain. However, it is found in the pre-mitotic microspore and in the mature pollen grain (Sanger, 1968). Osmiophilic bodies are also present in the cytoplasm of the microspore and the pollen grain. Judging from their appearance in osmium-fixed material, they probably contain lipids (Sanger, 1968) and are termed here lipid bodies. Angold (1968) reports the appearance of similar lipid droplets in the vegetative cytoplasm of bluebell pollen after the generative cell has been formed. In H. katherinae, as in bluebell pollen, these droplets ensheath the generative cell. In H. katherinae pollen, however, they are also present in the cytoplasm of the generative cell and are scattered throughout the vegetative cell as well. We thank Dr Peter K. Hepler for his suggestions and Dr Joseph W. Sanger for his assistance in various stages of this study. This investigation was supported by PHS Microbiology Training Grant 3 TO to the first author and by a grant to the second author from the Agriculture Research Service, U.S. Department of Agriculture, grant no (34) administered by Crops Research Division, Beltsville, Maryland. REFERENCES ANGOLD, R. E. (1968). The formation of the generative cell in the pollen grain of Endynrion non-scriptus (L). J. Cell Sci. 3, BAILEY, I. W. (i960). Some useful techniques in the study and interpretation of pollen morphology. J. Arnold Arbor. 41,

5 Fine structure of developing pollen 293 BOPP-HASSENKAMP, G. (i960). ElektronenmikroskopischeUntersuchungen an Pollenschlauchen zweier Lilaceen. Z. Naturf. B 15, DIERS, L. (1963). Electronenmikroskopische Beobachtungen an der generativen Zelle von Oenotliera hookeri Torr. et Gray. Z. Naturf. B 18, HESLOP-HARRISON, J. (1968). Synchronous pollen mitosis and the formation of the generative cell in massulate orchids. J. Cell Sci. 3, LARSON, D. A. (1965). Fine-structural changes in the cytoplasm of germinating pollen. Am. J. Bot. 52, LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biocfievi. Cytol. 9, MARUYAMA, K., GAY, H. & KAUFMANN, B. P. (1965). The nature of the wall between generative and vegetative nuclei in the pollen grain of Tradescantia paludosa. Am. J. Bot. 52, MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966). Cell division in the formation of the stomatal complex of young leaves of wheat. J. Cell Sci. 1, REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SANGER, J. M. (1968). An Ultrastructural Analysis of Pollen Development in Haemanthis katlierinae Baker. Ph.D. thesis, Dartmouth College. SASSEN, M. M. A. (1964). Fine structure of petunia pollen grain and pollen tube. Acta bot. veerl. 13, {Received 29 June 1970) irev cp dp f gc gch gw i I m IATIONS ON PLATES cell plate dividing plastid furrow generative cell generative chromatin generative wall intine lipid body mitochondrion mf mt n P pw V vc veil vn membrane fragments microtubule nucleus plastid pollen wall vacuole vegetative cell vegetative chromatin vegetative nucleus

6 294 J- M. Sanger and W. T. Jackson Fig. i. Mature microspore prior to cell division. Vacuoles (v) are positioned between the nucleus and the furrow (/). Plastids (p) usually contain starch (arrows), and lipid bodies (/) are distributed throughout the cytoplasm, x Fig. 2. Microspore during prophase of mitosis. Most of the organelles lie between the furrow and nucleus. The plastids sometimes contain osmiophilic droplets (arrows). The area in brackets is shown at higher magnification in Fig. 3. x 3700.

7 Fine structure of developing pollen

8 296 J. M. Sanger and W. T. Jackson Fig. 3. Higher magnification of the area in brackets in Fig. 2. Randomly arrayed microtubules (arrows) are present adjacent to the nucleus (;»). x Fig. 4. A plastid which seems to be dividing (dp) is present in the cytoplasm of a microspore undergoing mitosis, x Fig. 5. Telophase of microspore mitosis. Most of the organelles are between the furrow (/) and the cell plate (cp). A plastid which seems to be dividing (dp) is adjacent to the chromatin (vcfi) of the future vegetative cell. Mitochondria (in), but no plastids, are present between the cell plate and the chromatin (gch) of the future generative cell, x 4500.

9 Fine structure of developing pollen 297

10 298 J. M. Sanger and W. T. Jackson Fig. 6. Newly formed pollen grain. The generative cell wall (gw) is attached to the pollen wall opposite the furrow of the grain. Both nuclei are approximately spherical and of equal size, although in this figure they appear unequal in size due to plane of the section, x Fig. 7. Fusion of the generative (gw) and the pollen cell walls (pw). The generative wall is formed of finely fibrillar material (arrows) located between 2 unit membranes. Membrane fragments (inf) are located in the area of fusion of the generative wall with the intine (t) of the pollen wall, x

11 Fine structure of developing pollen 299

12 3<x> J. M. Sanger and W. T. Jackson Fig. 8. A high-magnification view of membrane fragments (mf) found in the area of fusion of the 2 walls, and the 2 unit membranes (arrows) adjacent to the generative wall. Microtubules (mf) are found in both the generative (gc) and vegetative (vc) cells in the area of fusion, x 75 coo. Fig. 9. Pollen grain soon after detachment of the generative cell. Lipid bodies (I) surround the generative cell and are found scattered in the cytoplasm of both cells. The vegetative nucleus (vn) is no longer spherical. There has been a great increase in the number of organelles within the cytoplasm of the vegetative cell, x 4500.

13 Fine structure of developing pollen

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