STUDIES ON THE FORMATION OF 'FLOATING' GUARD CELL MOTHER CELLS IN ANEMIA

Transcription

1 J. Cell Sri. 80, (1986) 29 Printed in Great Britain The Company of Biologists Limited 1986 STUDIES ON THE FORMATION OF 'FLOATING' GUARD CELL MOTHER CELLS IN ANEMIA B. GALATIS, P. APOSTOLAKOS AND D. PALAFOUTAS Institute of General Botany, University of Athens, GR , Athens, Greece SUMMARY The protodermal cells producing the 'floating' guard cell mother cells (GMCs) in three Anemia species undergo an extraordinary polarization and an unexpected shaping. During interphase an intercellular space is initiated at the internal proximal end of the cell, while the polar region bulges outwards. At this stage a microtubule girdle traverses the cortical cytoplasm underneath the rims of the external periclinal wall curvature. In addition, another system of microtubules converges on a cortical site adjoining the wall delimiting the intercellular space and, or, the neighbouring region of the internal periclinal wall (internal polar cortical site, IPCS). Vacuoles are found in all regions of the cell except for that between the centrally located nucleus and the intercellular space. As the cell approaches mitosis, the growing vacuolar system retreats from the cytoplasmic region below the external periclinal wall curvature. In most cells the polarized cytoplasm forms an inclined truncated cone, the bases of which abut on the external periclinal wall curvature and the wall lining the IPCS. The organization of the cortical microtubule cytoskeleton does not change significantly during preprophase-prophase. A preprophase microtubule band (PMB) is localized in the cortex lining the rims of the external periclinal wall curvature, while some microtubules traverse the IPCS and the cytoplasm adjacent to the neighbouring wall regions. The mitotic spindle axis is diagonal, while the cell plate separating the GMCs exhibits an unusual mode of growth. It gradually encircles the proximal daughter nucleus, becoming funnel-shaped. One of its periclinal edges fuses with the external periclinal wall area lined by the PMB cortical zone and the other with the internal periclinal wall area adjoining the IPCS. The latter region seems to behave like the PMB cortical zone. The results show that the morphogenetic mechanism underlying the formation of the conical GMCs includes a series of highly integrated processes, initiated or carried out during cell polarization. INTRODUCTION Among stomatal complexes, those of 'libera' (Prantl, 1881) or 'floating' (Mickel, 1962) stomata are unique. In surface view, they appear surrounded by an annular subsidiary cell, without coming into contact with any of its anticlinal walls. The floating stomata of Anemia were originally illustrated by Link (1841), while many authors have studied their ontogeny. Different hypotheses about guard cell mother cell (GMC) formation in Anemia have been proposed, among which two have prevailed (for reviews, see Mickel & Lersten, 1967; Probst, 1971; Fryns-Claessens & Van Cotthem, 1973). According to the first of them, a protodermal cell undergoes a periclinal division, which separates a small lens-shaped cell, the GMC, apposed on the external periclinal wall and a larger one that will become the subsidiary cell. The Key words: Anemia, guard cell mother cell formation, cell polarization, cortical microtubules, cell plate morphogenesis.

2 30 B. Galatis, P. Apostolakos and D. Palafoutas GMC gradually grows inwards up to the internal periclinal wall to which it fuses (Hildebrand, 1866; see also Oudemans, 1865). According to the other hypothesis, the GMC is formed in situ as a conical cell that never grows inwards (Rauter, 1870). Since then the development of Anemia stomata has been repeatedly considered. Among others, Probst (1971) and, particularly, Mickel & Lersten (1967), Humbert & Guyot (1969) and Gunning (1983) have supported Rauter's view. In contrast, Hildebrand's hypothesis has been more recently backed by Pant & Khare (1972) and particularly by Patel (1976), who discussed extensively GMC formation in Anemia. A detailed examination of the available literature reveals that the structural evidence presented to date is not adequate to substantiate either of the views. The present work re-examines the mode of GMC formation in three Anemia species using serial sectioning and correlated light and electron microscopy to study the morphogenetic mechanism of cell plate development. Particular attention has been paid to: (a) polarization of the protodermal cells as they prepare to produce the GMCs; (b) organization of a PMB in these cells; (c) prediction of the fusion sites of the cell plate with the parent walls by the PMB; and (d) conformational changes of the cytoplasm during cell polarization and division. The cytological parameters that must prevail in dividing higher plant cells in order that the cell plate can assume the predetermined shape and find its final position are more appreciable in curved cell plates than in planar ones (see Galatis, Apostolakos & Katsaros, 1983a, 1984a,fe). The protodermal cells producing the GMCs in Anemia represent a suitable cell type to justify further investigation of the above-mentioned phenomena. The mechanism (s) by which the plane of plant cell division is determined and accomplished are the subject of much current research (for a recent review, see Gunning, 1983; see also, Tiwari, Wick, Williamson & Gunning, 1984). MATERIALS AND METHODS Young leaflets of Anemia rotundifolia, Anemia mandiocanna and Anemia phyllitidis were fixed in 3 % glutaraldehyde in M-phosphate buffer, containing 1 % tannic acid or 1 % caffeine, for 3 6 h at room temperature, followed by 1 % OsO^ in the same buffer for 4 6 h at 0 C. Caffeine was added in the fixative in order to preserve the tannin-like inclusions in the vacuoles (Mueller & Greenwood, 1978). After dehydration with acetone, the specimens were infiltrated and embedded in Durcupan ACM (Fluka) or Epon 812, or Spurr'a mixture. Semi-thin sections were prepared from material fixed and embedded according to the above procedure. They were stained with 1 % Toluidine Blue in 1 % borax solution. Thin sections, double stained with uranyl acetate and lead citrate, were examined with a Philips 300 or a Hitachi HS-8 electron microscope. Electron micrographs of A. rotundifolia cells are presented because they were the most satisfactorily preserved of the Anemia species investigated. Some leaflets, after dehydration, were critical-point dried from liquid CO2, and attached to stubs. After sputter coating with gold, they were examined with a Cambridge S 150 scanning electron microscope. RESULTS Interphase initial stomatal cells On the lower leaflet surface of the Anemia species examined, stomata as well as hairs are formed (Fig. 1). The GMCs (Fig. 2) as well as the hair mother cells

3 Guard cell mother cell formation in Anemia 31 (Fig. 3) are produced in an acropetal gradient in the meristematic marginal leaflet area by asymmetrical differential divisions. In single paradermal sections it is not possible to recognize with any degree of certainty the interphase protodermal cells destined to produce the GMCs (initial stomatal cells; ISCs) (Fig. 3). Reliable criteria by which to distinguish them were found in transverse sections of leaflets made parallel to the veins. In those sections, the ISCs appear to undergo a unique and prolonged structural polarization. This process starts during interphase and is accompanied by a local change in cell shape. The proximal region of the cell, to which the future cell plate separating the GMC will anchor, bulges slightly but detectably outwards (Figs 4, 11, 12). Simultaneously, the junction of the internal periclinal wall with some of the anticlinal one(s) at the polar end of the cell is locally detached from the walls of the proximal protodermal and the underlying cell. In this way a small intercellular space appears, which gradually enlarges (Figs 4, 11, 12). The initial detachment of the walls seems to be the consequence of the shaping of the ISC and not of any of the adjacent protodermal cells. The initiation of the intercellular space at the polar endof the cell is a consistent phenomenon, always occurring long before the onset of mitosis. This can be used as a reliable criterion to identify the polarizing protodermal cells that will function as ISCs. Similar spaces do not develop at a proximal, distal or any other wall junction of protodermal cells dividing symmetrically in this protodermal area or those producing the hair mother cells (Fig. 36). Many ISCs appear to be derived from asymmetrical differential divisions. They differ in both size and fine structure from the daughter cells separated by the same divisions (Fig. 3). However, in other cases these differences are only slightly defined or absent. The interphase ISCs usually contain small vacuoles, which are found in all cytoplasmic areas except between the centrally positioned nucleus and the wall delimiting the intercellular space (Figs 3, 4, 5). In the latter region, as well as in the proximal cytoplasmic area, many mitochondria and some plastids are preferentially accumulated. Some of them are apposed to or lie in close proximity to the nuclear envelope (Fig. 4). A detailed examination of 20 interphase ISCs in serial sections revealed that the nucleus forms a local projection towards a cytoplasmic site adjacent to the wall outlining the intercellular space and, or, the neighbouring one of the internal periclinal wall (internal polar cortical site, IPCS). This projection, which is closely flanked by microtubules, approaches the plasmalemma (Figs 4, 9). A closer inspection of the interphase ISCs showed that the IPCS and the cytoplasm adjoining the neighbouring regions of the proximal anticlinal wall(s) and the internal periclinal one are traversed by a significant number of microtubules (Figs 8, 9, 11, 12l-o). In addition, some other microtubules running through inner cytoplasmic regions terminate in the IPCS (Figs 11, 12l-o). They are associated with plastids and mitochondria (see Fig. 16, and inset). Examination of serial paradermal and transverse sections shows that all the above microtubules, as well as those flanking the nuclear projection, converge on the IPCS (Figs 11, 12). Only a small number of microtubules exists along the distal and other anticlinal walls (Fig. 12). Occasionally, the cortical cytoplasm adjacent to the lower part of the proximal

4 32 B. Galatts, P. Apostolakos and D. Palafoutas

5 Guard cell mother cell formation in Anemia 33 Fig. 5. Diagrams made from serial semi-thin sections of an interphase ISC of A. mandiocanna to show the vacuolar distribution. anticlinal wall above the intercellular space is specified as an IPCS. This is shown by the convergence of microtubules and the protrusion of the nucleus towards this site (Fig. 10, and inset). In the interphase ISCs a large population of microtubules runs through the cytoplasmic cortex underlying the external periclinal wall. Most of them are arrayed in groups that line the rims of the external periclinal wall curvature, forming a ring (Figs 6, 7, 11, 12A D). The cortical cytoplasm adjacent to the external periclinal wall curvature is traversed by some microtubules, usually clustered in bundles. They appear to reach the microtubule ring but do not exhibit a definite orientation. Abbreviations used in figures: ch, chromosome; cp, cell plate; daw, distal anticlinal wall; ec, epidermal cell; epw, external periclinal wall; er, endoplasmic reticulum; gmc, guard cell mother cell; he, hair cell; hmc, hair mother cell; ipvi, internal periclinal wall; is, intercellular space; isc, initial stomatal cell; law, lateral anticlinal wall; m, mitochondrion; mt, microtubule; n, nucleus; ne, nuclear envelope;/), plastid;paw, proximal anticlinal wall; sc, subsidiary cell; st, stoma; v, vacuole. Fig. 1. Scanning electron micrograph of a protodermal area of the lower leaflet surface of A. mandiocanna. GMCs, differentiating stomata and hairs can be observed. The arrow points to a cell, which may represent an ISC. X650. Fig. 2. Paradermal view of a GMC and the surrounding subsidiary cell of A. rotundifolia. X3000. Fig. 3. Paradermal section of a protodermal area of A. rotundifolia, where stomatal formation starts. Two ISCs are depicted. One of them (arrow) has probably been derived from the asymmetrical differential division of a protodermal cell. X2300. Fig. 4. Transverse section of a polarized interphase ISC of A. rotundifolia. Note the outward bulging of the cell, the formation of an intercellular space at the polar end and the distribution of the vacuoles. The nucleus has formed a protrusion towards the cortical cytoplasm adjacent to the wall lining the intercellular space. X5000.

6 34 B. Galatis, P. Apostolakos and D. Palafoutas Finally, it must be noted that a number of microtubules traverse, singly or clustered in bundles, the perinuclear area. Preprophase initial stomatal cells As the ISC approaches mitosis, the protoplast organization changes further. Usually, the vacuoles, which have meanwhile grown, tend to fuse with each other to form one or few large vacuoles (Figs 13, 14). The vacuolar system retreats from the cytoplasmic area below the external periclinal wall curvature, while the cytoplasmic area between the nucleus and the IPCS remains free of vacuoles (Fig. 15). Reconstruction of the polarized cytoplasm shows that it takes the form of a more or less truncated inclined cone. This cytoplasmic cone is well delineated in ISCs of A. phyllitidis and A. mandiocanna, which enclose a well-developed vacuolar system. On one side this cone abuts on the external periclinal wall curvature, and on the other, on the internal periclinal wall portion covering the IPCS (Fig. 14). The nucleus is again in a more or less central position but lacks a projection (Figs 13, 14). Most of the organelles, particularly the plastids and mitochondria, are gathered in the polar region of the cell (Figs 15, 16). However, this structural polarization is less evident in some ISCs arising closer to the meristematic leaflet margin, which lack a developed vacuolar system (Fig. 37). In all Anemia species investigated here, groups of microtubules were found in the cortical cytoplasm adjoining the rims of the periclinal wall curvature of the preprophase prophase ISCs (Figs 17, 18). From the examination of serial sections it can be concluded that the above microtubules represent profiles of a microtubule ring apposed to the rims of the external periclinal wall curvature externally outlining the cytoplasmic cone (Figs 19, 20A H). This occupies the same cortical position as the interphase microtubule ring and can be considered a PMB (Figs 19, 20; compare with Figs 11, 12). An unexpected feature recorded in preprophase-prophase ISCs is the existence of a microtubule population in the IPCS and the neighbouring cortical cytoplasm of the internal periclinal wall and the proximal anticlinal one (Figs 16, 19). During interphase in these regions microtubules were also localized (Figs 16, 19; compare with Figs 11, 12). These microtubules are independent of those of the preprophase band. A few others, entering deeper into the cytoplasm and converging on the IPCS, were also present (Figs 16, 19). The above observations reveal that: (a) the ISCs possess a preprophase microtubule cytoskeleton organized differently from that described in other higher plant cells (among others, see Wick & Duniec, 1983, 1984; Gunning & Hardham, 1982; Gunning, 1983; Schnepf, 1984; Eleftheriou, 1985); and (b) the pattern of the cortical microtubule organization does not change markedly from interphase to preprophase prophase. As prophase progresses, the clear perinuclear cytoplasmic zone becomes conspicuous. One of the poles of the mitotic spindle is stabilized in a central cytoplasmic site at the upper proximal end of the cell while the other lies at the inner distal end of it. Therefore, the mitotic spindle is diagonally arranged and parallel to the leaf veins (Figs 21, 38). Its axis is not perpendicular to the PMB plane, as in most

7 Guard cell mother cell formation in Anemia 35 categories of dividing plant cells, but consistently forms an angle with it. In most ISCs the conical configuration of the cytoplasm becomes more conspicuous during mitosis and early cytokinesis, occupying the same cellular domain (Figs 21, 22). Cytokinetic initial stomatal cells During mid-to-late anaphase, a typical cell plate starts being organized in a median plane between the daughter nuclei, transversely to the mitotic spindle axis (Figs 23 (inset), 29). In ISCs, the initial cell plate portion does not lie on the PMB plane or on a parallel one, but forms a definite angle with it (Fig. 23 (inset); compare with Fig. 19). The external margin of the initial cell plate portion, when it emerges from the internuclear area, grows, straight on an inclined plane, enters a distal site of the PMB cortical zone and fuses with the underlying wall (Fig. 23 (inset); see also Figs 26, 39). The opposite edge is directed to a distal region of the IPCS where it coalesces with the adjacent wall area (Fig. 23 (inset); see also Figs 26, 39). At this stage the anticlinal cell plate margins approach the vacuolar system (Figs 22, 29). Afterwards, the cell plate, expanding laterally, curves around the proximally positioned daughter nucleus and becomes funnel-shaped (Figs 30, 31, 32; see also Fig. 26). The external cell plate edge outlines a circular path, following with accuracy the PMB cortical zone and intersects the adjacent periclinal wall (Figs 30, 31; see also Fig. 26). In contrast, the internal margin traces a short path and joins the wall lined by the IPCS (see Figs 26, 39). This seems to follow the cytoplasmic area above the IPCS, which remains free of vacuoles during cell polarization and division. The anticlinal edges of the cell plate fuse with each other. This is initially achieved in Figs 6, 7. Details of the cortical cytoplasmic regions adjacent to the external periclinal wall areas, which are indicated by the brackets in the ISC illustrated in Fig. 4. The arrows point to microtubules. Fig. 6, X43 000; Fig. 7, X Fig. 8. The IPCS of the ISC depicted in Fig. 4 where the nucleus projects. This area is traversed by numerous microtubules (arrows) exhibiting different orientations, among which membranous elements are localized. Some of the microtubules are arranged in bundles. X Fig. 9. Portion of a transversely sectioned ISC of A. rotundifolia, including the IPCS and the nuclear projection towards it. Microtubules (arrows) flank the nuclear projection. X Fig. 10. Portion of a transversely sectioned ISC of A. rotundifolia, in which the cortical cytoplasm adjacent to the proximal anticlinal wall above the intercellular space, which is very small, seems to function as an IPCS. X The inset depicts the ISC, part of which is shown in Fig. 10. X1500. Fig. 11. Diagrams made from transverse sections of the interphase ISC of A. rotundifolia illustrated in Fig. 4, showing the microtubule organization and distribution in four planes. The dots denote transversely sectioned microtubules; the lines, longitudinally sectioned ones. The perinuclear microtubules were not drawn. Fig. 12. Tracing of cortical microtubules as well as those flanking the nuclear projection in successive paradermal planes of an A. rotundifolia ISC. The microtubules lining the external periclinal wall form a ring (A-D), while those lining the internal periclinal wall and the proximal anticlinal wall region as well as the others flanking the nuclear projection converge on the IPCS (G O). The dots and the lines denote transversely and longitudinally sectioned microtubules.

8 36 B. Galatis, P. Apostolakos and D. Palafoutas wmmm X «*** is Figs For legends see p. 35

9 Guard cell mother cell formation in Anemia 37 daw epw 12 K L M N Figs For legends see p. 35

10 38 B. Galatis, P. Apostolakos and D. Palafoutas the mid-plane of the cell (Figs 31G,H). At the advanced stages of cell plate growth numerous mitochondria are gathered in the area where the anticlinal cell plate margins will fuse with each other (Fig. 32). In internal paradermal planes the posttelophase GMCs exhibit variable profiles, in contrast to the external ones in which Figs 13, 14. Diagrams made from serial paradermal (Fig. 13) and transverse (Fig. 14) semi-thin sections of an A. mandiocanna and an A. rotundifolia preprophase ISC, respectively. The vacuole organization and development differs from those of the interphase ISCs (compare with Fig. 5). The polar cytoplasm tends to assume the form of a truncated cone. Fig. 15. Median transverse section of a preprophase ISC of A. rotundifolia. X3800. Fig. 16. Higher magnification of the IPCS and the neighbouring cytoplasmic regions of the preprophase ISC shown in Fig. 15. Note the mitochondria accumulation and the significant number of microtubules (arrows) traversing and converging on the IPCS. The mitochondria exhibit associations to microtubules. X The inset shows details of a mitochondrion-microtubule association. X Figs 17, 18. Enlargement of the PMB cortical zone profiles defined by the dark lines in the ISC shown in Fig. 15. Two microtubule groups (arrows) representing sections of a PMB are observed. Fig. 17, X40000; Fig. 18, X40000.

11 Guard cell mother cell formation in Anemia i?.«.i< WrfL^mhf*

12 40 B. Galatis, P. Apostolakos and D. Palafoutas epw 20 H paw K Figs 19, 20. Diagrams to trace the microtubules of two preprophase ISCs of A. rotundifolta in selected transverse (Fig. 19) and paradermal (Fig. 20) sections. In both cases the perinuclear microtubules have not been drawn. The transversely and longitudinally sectioned microtubules are shown by dots and lines, respectively.

13 Guard cell mother cell formation in Anemia 41 B D 22 Fig8 21, 22. Diagrammatic representation of a mitotic (Fig. 21) and an early cytokinetic (Fig. 22) ISC of A. mandiocanna made from serial paradermal semi-thin sections. Note the configuration of the polar cytoplasm and of vacuolar system. they always show annular or ovoid profiles (Fig. 35; compare with Figs 2, 42). Occasionally, the internal end of the cell plate fuses at a region of the lower part of the proximal anticlinal wall above the intercellular space (Fig. 28, and inset). In these cells the IPCS is probably localized in the above-mentioned cortical area (see Fig. 10, and inset). In the ISC illustrated in Fig. 46 the intercellular space has not been formed at the wall junction, which is proximal to the leaflet margin, but at a polar region of a lateral wall junction (Fig. 47). The internal end of the cell plate meets the older wall lining this area. A cytokinetic event deserving attention is the formation of numerous associations between the phragmoplast microtubules and the GMC daughter nucleus (Fig. 23). The former attach to the nuclear envelope (Figs 24, 25). In these regions the nucleus forms slight depressions or short protrusions towards the cell plate (Figs 24, 25).

14 42 B. Galatis, P. Apostolakos and D. Palafoutas In contrast, relatively few associations were observed between the subsidiary cell nucleus and the phragmoplast microtubules, although the distance between the cell plate and both the nuclei is the same (Figs 23 (inset), 29). Associations between the GMC nucleus and the phragmoplast microtubules are observed during all stages of cell plate growth (Figs 32 (inset), 33). During cytokinesis the GMC daughter nucleus becomes conical (see Figs 34, 39). Initially, in median planes, this exhibits a U shape or a deep invagination, while its internal part is slender (Figs 26, 30, 32). Phragmoplast microtubules are associated with the nuclear lobes (Figs 32 (inset), 33). Microtubules are also found in the cytoplasmic pocket formed by the GMC nucleus (Fig. 27). A small number of GMCs in all Anemia species examined are separated by an anticlinally oriented and intensely curved cell plate, the anticlinal margins of which fuse with the proximal anticlinal wall(s) of the parent cell (see Fig. 41). These GMCs are the first-formed in the leaflets. The ISCs giving rise to the above GMCs were not identified for study. Sometimes the internal part of the GMC is bridged by the proximal anticlinal wall of the subsidiary cell with a short piece of wall (Fig. 43). This wall is included in a few paradermal sections (see Fig. 42) and is formed by the asymmetrical growth of the curving edges of the cell plate. Probably, for the same reason, some GMCs do not reach the internal periclinal wall, but are connected with it by a short wall (Fig. 40). The latter represents the daughter wall region laid down by the initial portion of the cell plate separating the GMCs. The GMCs gradually enlarge and change in shape. The intercellular space extends below the GMC and the subsidiary cell, becoming a typical substomatal cavity (Fig. 34). The internal anticlinal wall portion of the young GMCs is detached from its partner wall of the subsidiary cell (arrows in Fig. 34). The GMCs grow further outwards and become raised above the epidermis (Fig. 34; see also Fig. 1). The lower part of the cell enlarges to some extent but the cell retains its conical form. Finally, it must be noted that the highly vacuolated subsidiary cells may also enter mitosis. The nucleus divides in a distal position of the cell close to the GMC or the young stoma (Fig. 44). The cell plate connects the distal pole of the stoma or the GMC with a distal region of the subsidiary cell wall (Fig. 45). This cell remains incompletely divided. DISCUSSION General remarks on GMC development In order to explain the conflicting views on the development of the peculiarly shaped GMCs of the 'floating' stomata of Anemia, the supporters of Hildebrand's hypothesis maintain that Rauter (1870) and other investigators sharing his opinion have missed the very early stages of GMC development (see Patel, 1976). On the other hand, the followers of Rauter's view consider the lens-shaped GMCs, which have been described as newly formed GMCs, to be eccentric profiles of conical ones (see Mickel & Lersten, 1967).

15 Guard cell mother cell formation in Anemia 43 The detailed examination of numerous cytokinetic ISCs in three Anemia species shows unequivocally that they are separated by a funnel-like cell plate. After prolonged and complicated growth the periclinal cell plate edges intersect particular regions of the periclinal walls, while the anticlinal ones fuse with each other (Figs 30, 31). The controversy concerning GMC formation, among other reasons, probably arose from the fact that the internal part of the newly formed GMC is very thin. Sometimes it may be included in two or three transverse semi-thin sections. In all other transverse sections it exhibits a more or less lens-shaped form. Therefore, Rauter's view of GMC development in Anemia is correct. It was also found that the anticlinal walls of some stomata bridging the distal stomatal pole with the neighbouring anticlinal wall region of the subsidiary cell are formed by incomplete divisions of the latter cell type. Therefore, the suspended Anemia stomata described by Strasburger ( ) and Prantl (1881) initially develop as floating stomata and are connected later with the subsidiary cell wall. The substomatal intercellular space formation is a well-known event in stomatal ontogeny. Pfitzer (1870), for instance, has described the stomata as originating from protodermal files by the development of substomatal chambers below the future GMCs. However, the initiation of the substomatal cavity in the polar area of the Anemia ISCs and its relationship to the polarization sequence was noted for the first time here (see next section). Polarization and shaping of initial stomatal cells The GMC in Anemia is separated by an asymmetrical differential division characterized by a unique polarity. The polarized cytoplasm forms a more or less inclined truncated cone in the proximal end of the cell, a phenomenon made evident Fig. 23. Transverse view of a growing cell plate in an early cytokinetic ISC of A. rotundifolia. Associations between the phragmoplast microtubules and the GMC daughter nucleus have been developed (arrows), x The inset shows the ISC, the cell plate of which is illustrated in Fig. 23. The initial cell plate portion following a straight path enters a distal site of the PMB cortical zone and of the IPCS (arrows). X1300. Figs 24, 25. Higher magnification of the cytokinetic ISC areas shown in Fig. 23 (inset), taken from adjacent sections. Details of the associations between the phragmoplast microtubules and the nuclear envelope are evident. Fig. 24, X45 000; Fig. 25, X Fig. 26. Slightly eccentric transverse section of an advanced cytokinetic ISC of A. rotundifolia. The arrowheads define the cell plate. The growing cell plate edge locally diverges to meet the PMB cortical zone (arrow). Note the fusion site of the distal cell plate edge with the external periclinal wall (arrow), the vacuolar disposition, the GMC nuclear shape and the distribution of plastids and mitochondria in the cell. X7000. Fig. 27. Higher magnification of the cytoplasmic pocket formed by the GMC nucleus in the cytokinetic ISC shown in Fig. 26. Microtubules enter the nuclear cavity. X Fig. 28. Portion of a young GMC of A. rotundifolia, the internal end of which has fused with the proximal anticlinal wall above the intercellular space (arrows). X The inset illustrates the GMC, part of which is shown in Fig. 28. X1600. Fig. 29. An early cytokinetic ISC of A. rotundifolia in a median paradermal view. At this stage the slightly curved cell plate has reached the vacuolar system. Many mitochondria, as well as plastids are concentrated in the proximal end of the cell. X6000.

16 44 B. Galatis, P. Apostolakos and D. Palafoutas Figs For legends see p. 43

17 Guard cell mother cell formation in Anemia epw s \ 28, ^f'^' Figs For legends see p. 43

18 46 B. Galatis, P. Apostolakos and D. Palafoutas K 31 G HI j Figs 30, 31. Diagrammatic paradermal representation of two late cytokinetic ISCs of A. mandiocanna. Note the vacuole arrangement and the curved growth of the cell plate in different planes. In Fig. 31 the fusion of the anticlinal cell plate edges has been completed first in a median plane of the cell (G,H). by the shaping of the vacuolar system. The nucleus and the other organelles occupy definite domains in the polarized cytoplasm, while the cell possesses a particularly organized microtubule cytoskeleton. The question arising, then, is why is the polarization of the Anemia ISCs so different from that established in the dividing stomatal cells of angiosperms (among others, see Biinning & Biegert, 1953; Stebbins & Shah, I960; Pickett-Heaps & Northcote, 1966; Galatis & Mitrakos, 1979; Busby & Gunning, 1980). Relying on the present state of knowledge it is not possible to follow the primary steps of cell polarization and the concomitant qualitative changes of the cytoplasm. However, in Anemia ISCs among the first structural, and probably causal, events of

19 Guard cell mother cell formation in Anemia 47 polarization is a definite change in the relative directions of cell growth, which results in the initiation of the intercellular space and the outward bulging of the cell (Fig. 4). This shaping reflects pictorially a morphogenetic phenomenon that has not been described in stomatal cells of any other plant. The relationship between cell shaping and polarization is expressed by (1) the orientation of the truncated cytoplasmic cone and (2) the particular organization of cortical microtubules. In our opinion, the Fig. 32. Portion of a paradermal section of a late cytokinetic ISC including the growing cell plate (arrowheads). The GMC nucleus exhibits a lobed appearance, while plastids and mitochondria are gathered in the proximal end of the cell. The mode of cell plate growth can be followed (arrowheads). X In the inset the phragmoplast microtubules from five serial sections of an ISC, one of which is Fig. 32, were traced. The microtubules (lines) connecting the cell plate edges with the GMC nucleus form bundles attached to the nuclear envelope in deepenings of its lobes. Fig. 33. Details of the microtubule association between the cell plate margin shown by the arrow in the ISC in Fig. 32 and the GMC daughter nucleus. The microtubules (arrow) come into contact with the nuclear envelope. X Fig. 34. Median transverse section of a differentiated GMC of A. rotundifolia. The intercellular space has been expanded into a substomatal cavity, while the GMC has been raised over the epidermis. X35OO. Fig. 35. Paradermal view of the internal wall portion of a post-telophase GMC of A. rotundifolia. This is connected to the proximal anticlinal wall by a short piece of wall (arrow). X8500. Fig. 36. Growing hair-mother cell of A. rotundifolia in a transverse section. An intercellular space has not been developed in the junction of the internal periclinal wall with any of the anticlinal ones and the adjacent walls of the neighbouring cells. X1000. Fig. 37. A mitotic ISC of A. pkyllitidis as it appears in a paradermal plane. This cell lies close to the meristematic leaflet margin and lacks a developed vacuolar system. X Fig. 38. A metaphase ISC of A. rotundifolia as it appears in a median transverse section. Note the ISC shape and the orientation of the chromosome plate. X1000. Fig. 39. Median transverse section of a typical conical GMC of A. rotundifolia. Note the fusion sites of the GMC wall with the parent periclinal walls (arrows) and the shape of the GMC nucleus. X1000. Fig. 40. An atypical GMC of A. rotundifolia. The internal cell plate edges have fused with each other. The GMC comes into contact with the internal periclinal wall of the parent cell by a short piece of wall (arrow). X1000. Fig. 41. Paradermal view of an A. pkyllitidis stoma formed in contact with the proximal anticlinal wall of the parent cell (applied stoma). X800. Figs 42, 43. Paradermal sections of a GMC of A. mandiocanna. The internal portion of the cell is connected to the proximal anticlinal wall by a short piece of wall (see arrow in Fig. 43) not seen in Fig. 42. Fig. 42, X800; Fig. 43, x800. Fig. 44. Prophase subsidiary cell of A. mandiocanna. X800. Fig. 45. Stoma of A. mandiocanna. The distal pole of this stoma is connected with the distal region of the subsidiary cell with a wall (arrow) laid down during division of the subsidiary cell. Compare with the prophase subsidiary cell shown in Fig. 44. X800. Figs 46, 47. Paradermal sections of an A. mandiocanna GMC. In this case the intercellular space (see arrow in Fig. 47) has been developed at the junction of a lateral anticlinal wall with the internal periclinal one and the adjacent walls of the neighbouring cells. Fig. 46, x800; Fig. 47, X800.

20 48 B. Galatis, P. Apostolakos and D. Palafoutas Figs For legends see p. 47

21 Guard cell mother cell formation in Anemia Figs For legends see p. 47

22 50 B. Galatis, P. Apostolakos and D. Palafoutas relationship between the local cell shaping and polarization, expresses a 'morphogenetic shift* of the ISC protoplast that, through the establishment of polarity, triggers a particular sequence of interrelated phenomena. The microtubular cytoskeleton, among other probable functions, may play a role in the determination of the pattern of the 'morphogenetic shift' and the spatial organization of the protoplast. Some of the polarity phenomena and cytoplasmic parameters determining or affecting the funnel-like pattern of cell plate growth are discussed below. Cytoplasmic configuration in polarized initial stomatal cells The shaping of the large vacuolar system, which accompanies the formation of a stable cytoplasmic cone in ISCs, presupposes a highly controlled reorganization of the cytoplasmic matrix. This process can be easily understood if the latter is organized as a microtrabecular lattice as has been observed in some animal and plant cell types (Porter & Tucker, 1981; McNiven & Porter, 1984; Wardrop, 1983; Cox & Juniper, 1983; Hawes, Juniper & Home, 1983). At this point our interest is focused on the question: what is the real significance of the cytoplasmic configuration in the ISCs in cell plate morphogenesis? Although the polarized nature of the cytoplasmic matrix and the cytoplasmic basis of polarity have been appreciated long ago (see Frey-Wyssling, 1948; Bunning, 1952; Bloch, 1965), the behaviour of the cytoplasm during polarization and cell division usually escapes the attention of the investigators. An exception to this is the observation of the phragmosome in highly vacuolated cells, i.e. of a fenestrated cytoplasmic baffle on the plane of cell division in which mitosis and cytokinesis are carried out (Sinnott & Bloch, 1940, 1941). Such a baffle may be organized in the lessvacuolated plant cells (Gunning, 1983; see also Brown & Lemmon, 1984, 1985), but it is not outlined. The truncated cytoplasmic cone organized in the ISCs can be considered as an unusual type of phragmosome. Its shape roughly resembles that of the GMC. The fact that in some ISCs lacking a developed vacuolar system the cytoplasmic cone is not evident does not rule out the possibility of cytoplasmic reorganization. Although in this cone the nucleus divides and the cell plate is organized, it does not seem likely that it is able to pattern the funnel-shape of the cell plate. Unless the nucleus occupies a specific domain in the ISC and the mitotic spindle axis has a specific diagonal alignment, this cell plate shape cannot be attained. The above cellular parameters ensure that the initial cell plate portion growing straight will encounter a distal site of the PMB cortical zone and the IPCS (Fig. 23 (inset); compare with Fig. 19). After consolidation of the initial cell plate portion and the determination of its direction of growth, the PMB cortical zone, the IPCS and the cytoplasmic cone seem to define the curved path followed by the cell plate. Following the above path, the anticlinal cell plate edges become exactly opposite and fuse with each other (Figs 30, 31). This suggests an interaction between these margins, probably carried out by the phragmoplast microtubules. However, the phenomenon is far from being considered solved. Unknown activities of the polar cytoplasm may be involved.

23 Guard cell mother cell formation in Anemia 51 Specification of the cortical sites of cytokinesis One of the new findings of the present work is that the fusion sites of the funnellike cell plate with the parent walls are predictable from a preprophase-prophase stage. They are the periclinal wall portions lined by the PMB cortical zone and the IPCS. The external periclinal edge of the cell plate follows exactly the PMB cortical zone, while the internal one fuses with the wall area in which a microtubule population converged during interphase and preprophase prophase. These microtubules cannot be considered a PMB or a PMB portion. They do not exhibit an organization (Fig. 19) or a positional relationship to the cell plate resembling those of a PMB. Therefore, in Anemia ISCs, the PMB partly anticipates the fusion sites of the cell plate with the older walls. A partial inconsistency between the PMB and the final cell plate arrangement has been encountered in some protodermal cell types of Triticum (Galatis et al. 1983a, 1984a,6). In those cells the disturbance of premitotic polarity and, or, space limitations have been implicated as the factors responsible for cell plate divergence. This is expressed by the disturbance of the mutual disposition between the preprophase nucleus and the PMB as well as between the PMB cortical zone and the mitotic spindle. A difference between Triticum protodermal cells and Anemia ISCs is that in the latter cell type the PMB is committed to forecast one of the attachment sites of the cell plate with the older wall. A typical PMB totally organized in the cytoplasmic cortex below a periclinal wall of a protodermal cell is recorded for the first time here. This PMB arrangement, as well as the mitotic spindle alignment, initially has results in a limited portion of the cell plate edge entering the PMB cortical zone. Afterwards, the same edge outlining a circular path follows the PMB cortical zone and fuses with the rims of the external periclinal wall curvature. The observations presented in this article also reveal, for the first time in vascular plants, that in Anemia ISCs, apart from the PMB cortical zone, another region of the cortical cytoplasm, namely the IPCS, seems to be functionally differentiated so as to behave like the PMB cortical zone. A cortical cytoplasmic region, lying far from the PMB cortical zone and having the ability to control the direction of the cell plate growth has been found in some protodermal cell types of Marchantia paleacea among which intercellular spaces develop (Apostolakos & Galatis, 1985c). In the same cells incomplete PMBs are organized (Apostolakos & Galatis, 19856). In Anemia species examined here as well as in Marchantia paleacea, the cortical sites appearing to function like the PMB cortical zone underlie developing intercellular spaces. In those cortical regions of M. paleacea during interphase, as well as preprophase-prophase, it has been suggested that MTOCs operate (Apostolakos & Galatis, 1985a,6). inanemia interphase ISCs, MTOCs are probably activated in the IPCS. The following indications favour this hypothesis: (a) the convergence of many microtubules (Figs 11, 12), and (b) the projection of the interphase nucleus and the accumulation and, or, convergence of some plastids and mitochondria on the IPCS. Similar cytoplasmic events characterize the activation of prominent cortical MTOCs suggested as functioning in: (1) the GMCs of Zea mays (Galatis, 1982), (2) the differentiating guard cells of Adiantum capillus veneris (Galatis et al ) and

24 52 B. Galatis, P. Apostolakos and D. Palafoutas (3) some protodermal cells of Marchantia paleacea gametophyte (Apostolakos & Galatis, 1985a). In the latter cells, as well as in Anemia ISCs, the above MTOCs seem to remain active even during preprophase-prophase, nucleating and, or, organizing or maintaining a microtubule system independent of the PMB. In the interphase ISCs of Anemia cortical MTOCs also seem to function in the cytoplasm abutting the rims of the external periclinal wall curvature. Considering the microtubule organization in the above wall at interphase and preprophase prophase, it seems likely that either two sequential microtubule rings are nucleated and, or, organized by MTOCs functioning in the above sites, or one ring is formed during interphase, which later functions as a PMB. In the latter case, the interphase microtubule ring might represent an early stage of PMB formation. Long-lived PMBs have already been found by Schnepf (1973) and Gunning, Hardham & Hughes (1978). The local deformation of the cytoplasmic cortex, including the adjacent plasmalemma portions, which occurs during formation of the external periclinal wall curvature and the extension of the wall delimiting the intercellular space in the shaping ISCs, is a probable MTOC activator. Cortical MTOCs also appear to be activated in the locally expanding region of GMCs oizea mays (Galatis, 1982) and in some protodermal cells of M. paleacea (Apostolakos & Galatis, 1985a). GMC nucleus phragmoplast microtubule associations It is commonly held that, among other functions, the phragmoplast prevents the daughter nuclei from approaching and fusing with each other. However, in Anemia ISCs the fact that the phragmoplast microtubules form more associations with the nucleus that will be included in the GMC than with that of the separating subsidiary cell, suggests the existence of a preferential affinity between them and that these associations must have a particular significance. About the latter problem, only hypotheses can be offered. For instance, the formation of a small conical GMC at a particular region of the ISC presupposes that during cytokinesis the GMC daughter nucleus will occupy a specific domain. The cell plate is laid down closely around the nucleus, while its diameter decreases as it approaches the internal periclinal wall. Therefore, the GMC nucleus, in order to fit into the separating GMC must change shape locally. The GMC daughter nucleus-phragmoplast microtubule associations may be responsible for or related to the stabilization and shaping of the nucleus. Another possibility is that the above associations are involved in or are responsible for the curved growth of the cell plate. However, this attractive hypothesis cannot explain a number of phenomena such as: (a) the shaping of the telophase GMC nucleus; (b) the decrease of the cell plate diameter as it grows inwards; and (c) why the cell plate does not completely surround the nucleus, but assumes a funnel-like form. In addition, there is some evidence from previous work that the curved mode of cell plate growth during formation of the graminaceous subsidiary cells is not controlled by the subsidiary cell daughter nucleus (Galatis et al. 1983a, 1984a,6).

25 Guard cell mother cell formation in Anemia 53 Conclusions on the morphogenesis of the funnel-like cell plate Summing up the above discussion, it becomes evident that the morphogenesis of the funnel-like cell plate of Anemia ISCs is accurately controlled. The mechanism is the integration of a sequence of interrelated sub-processes, among which the following are the most important, (a) The specification of two cortical cytoplasmic regions independent of each other (PMB cortical zone and IPCS), where the cell plate will intersect the parent walls. MTOCs seem to operate in the above regions, (b) The particular diagonal orientation of the mitotic spindle, which determines that the cell plate at a specific angle will find a distal region of the PMB cortical zone and the IPCS. (c) The highly controlled spatial organization of the protoplast, which is expressed by shaping the vacuole(s) and the highlight of which is the formation of a particular cytoplasmic configuration, (d) The stabilization of the GMC daughter nucleus at a definite position, as well as its shaping so that it can be included and fit into the GMC. The first three processes start or take place during cell polarization, enabling the cell plate to follow the funnel-like path. The highly integrated character of the phenomenon is underlined by the fact that the functionally differentiated regions of the cell cortex are those abutting on the cytoplasmic cone. The authors thank Dr C. Fasseas for providing scanning electron microscope facilities. Living material of the Anemia species examined in this study was kindly supplied by the Botanical Garden of Miinchen-Nymphenburg. This work was supported by a grant from the Ministry of Research and Technology (no. 4.10/1985). REFERENCES APOSTOLAKOS, P. & GALATIS, B. (1985a). Studies on the development of the air pores and air chambers of Marchantia paleacea. II. Ultrastructure of the initial aperture formation with particular reference to cortical microtubule organizing centres. Can.jf. Bot. 63, APOSTOLAKOS, P. & GALATIS, B. (1985ft). Studies on the development of the air pores and air chambers of Marchantia paleacea. III. Microtubule organization in preprophase-prophase initial aperture cells - formation of incomplete preprophase microtubule bands. Protoplasma 128, APOSTOLAKOS, P. & GALATIS, B. (1985C). Studies on the development of the air pores and air chambers of Marchantia paleacea. IV. Cell plate arrangement in initial aperture cells. Protoplasma 128, BLOCH, R. (1965). Polarity gradients in plants: A survey. In Handbuch der Pflanzenphysiologie, 15/1 (ed. W. Ruhland), pp Berlin, Heidelberg, New York: Springer-Verlag. BROWN, R. C. & LEMMON, B. E. (1984). Plastid apportionment and preprophase microtubule bands in monoplastidic root meriatem cells of hoe tes and Selaginella. Protoplasma 123, BROWN, R. C. & LEMMON, B. E. (1985). Preprophasic establishment of division polarity in monoplastidic mitosis of hornworts. Protoplasma 124, BONNING, E. (1952). Morphogenesis in plants. Surv. Biol. Progr. 2, BUNNING, E. & BIEGERT, F. (1953). Die Bildung der Spaltoffnungsinitialen bei Allium cepa. Z.Bot. 41, BUSBY, C. H. & GUNNING, B. E. S. (1980). Observations on preprophase bands of microtubules in uniseriate hairs, stomatal complexes of sugar cane, and Cyperus root meristems. Eur. J. Cell Biol. 21, Cox, G. & JUNIPER, B. E. (1983). High-voltage electron microscopy of whole critical-point dried plant cells- Fine cytoskeletal elements in the moasbryum tenuisetitm. Protoplasma 115, ELEFTHERIOU, E. P. (1985). Microtubules and root protophloem ontogeny in wheat. J. Cell Set. 75,

26 54 B. Galatis, P. Apostolakos and D. Palafoutas FREY-WYSSLING, A. (1948). Submicroscopic Morphology of Protoplasm and its Derivatives. New York: Elsevier Publishing Company. FRYNS-CLAESSENS, E. & VAN COTTHEM, W. (1973). A new classification of the ontogenetic types of stomata. Bot. Rev. 39, GALATIS, B. (1982). The organization of microtubules in guard cell mother cells of Zea mays. Can. J. Bot. 60, GALATIS, B., APOSTOLAKOS, P. & KATSAROS, CHR. (1983a). Synchronous organization of two preprophase microtubule bands and final cell plate arrangement in subsidiary cell mother cells of some Triticum species. Protoplasma 117, GALATIS, B., APOSTOLAKOS, P. & KATSAROS, CHR. (19836). Microtubules and their organizing centres in differentiating guard cells oiadiantum capillus veneris. Protoplasma 115, GALATIS, B., APOSTOLAXOS, P. & KATSAROS, CHR. (1984a). Positional inconsistency between preprophase microtubule band and final cell plate arrangement during triangular subsidiary cell and atypical hair cell formation in two Triticum species. Can.jf. Bot. 62, GALATIS, B., APOSTOLAKOS, P. & KATSAROS, CHR. (19846). Experimental studies on the function of the cortical cytoplasmic zone of the preprophase microtubule band. Protoplasma 121, GALATIS, B. & MITRAKOS, K. (1979). On the differential divisions and preprophase microtubule bands involved in the development of stomata of Vigna sinensis. J. Cell Sci. 37, GUNNING, B. E. S. (1983). The cytokinetic apparatus: Its development and spatial regulation. In The Cytoskeleton in Plant Growth and Development (ed. C. W. Lloyd), pp London, New York: Academic Press. GUNNING, B. E. S. &HARDHAM, A. R. (1982). Microtubules. A. Rev. Plant Physiol. 33, GUNNING, B. E. S., HARDHAM, A. R. & HUGHES, J. E. (1978). Pre-prophase bands of microtubules in all categories of formative and proliferative cell divisions in Azolla roots. Planta 143, HAWES, C. R., JUNIPER, B. E. &HORNE, J. C. (1983). Electron microscopy of resin-free sections of plant cells. Protoplasma 115, HiLDEBRAND, F. (1866). Ueber die Entwickelung der Farnkrautspaltoffnungen. Bot. Ztg 24, HUMBERT, C. & GUYOT, M. (1969). Le d6veloppement des stomates ad6tostomes $ Anemia rotundifolia Schrad. (Famille des Schiz6ac6es). C.r. hebd. Seanc Acad. Sci. Paris, ser. D, 268, LINK, H. F. (1841). Icones selectae anatomico-botanicae... Ausgewahlte anatomisch-botanische abbildungen. Heft III, Taf. IV. Berlin: Haude and Spenersche. McNrvEN, M. A. & PORTER, K. R. (1984). Chromatophores - Models for studying cytomatrix translocations. J. Cell Biol. 99, 152s-158s. MICKEL, J. T. (1962). A monographic study of the fern genus Anemia, subgenus Coptophyllutn. Iowa State J. Sci. 36, MICKEL, J. T. & LERSTEN, N. R. (1967). Floating stomates (adetostomy) in ferns: Distribution and ontogeny. Am.J. Bot. 54, MUELLER, W. C. & GREENWOOD, A. D. (1978). The ultrastructure of phenolic-storing cells fixed with caffeine. J. exp. Bot. 29, OUDEMANS, C. A. J. A. (1865). Sur l'origine des stomates de quelque especes d'aneimia. Bull. Congr. Bot. et d'hort., Amsterdam, pp PANT, D. D. & KHARE, P. K. (1972). Epidermal structure and stomatal ontogeny of Anemia spp. Ann. Bot. 36, PATEL, J. D. (1976). Floating stomate - its probable ontogeny. Flora 165, PFTTZER, E. (1870). Beitr3ge zur Kenntnis der Hautgewebe der Pflanzen. Jb. toiss. Bot. 7, PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966). Cell division and the formation of the stomatal complex of the young leaves of wheat. J'. Cell Sci. 1, PORTER, K. R. & TUCKER, J. B. (1981). The ground substance of the living cell. Scient. Am. 244, PRANTL, K. (1881). Untersuchungen zur Morphologie der Gefdsskryptogamen. II. Die Schizaeaceen. Leipzig: Wilhelm Engelmann. PROBST, W. (1971). Vergleichende Morphologie und Entwicklungsgeschichte der Spaltoffnungen bei Farnen. Dissertation, Stuttgart.

27 Guard cell mother cell formation in Anemia 55 RAUTER, J. (1870). Entviicklungsgeschichte der Spaltoffnungen von Aneimia und Niphobolus. Mitteil. Naturwiss. Verein. Steiermark Band II, Heft II, pp ScHNEPF, E. (1973). Mikrotubulus-Anordnung und -Umordung Wandbildung und Zellmorphogenese in jungen Sp/ia^nMm-blattchen. Protoplasma 78, SCHNEPF, E. (1984). Pre- and postmitotic reorientation of microtubule arrays in young Sphagnum leaflets: Transitional stages and initiation sites. Protoplasma 120, SINNOTT, E. W. & BLOCH, R. (1940). Cytoplasmic behavior during division of vacuolate plant cells. Proc. natn.acad. Sci. U.SA. 26, SINNOTT, E. W. & BLOCH, R. (1941). Division of vacuolate plant cells. Am. J. Bot. 28, STEBBINS, L. G. & SHAH, S. S. (I960). Developmental studies of cell differentiation in the epidermis of monocotyledons. II. Cytological features of stomatal development in the Gramineae. Devi Biol. 2, STRASBURGER, E. ( ). Ein Beitrag zur Entwicklungsgeschichte der Spaltoffnungen. Jb. wiss. Bot., Pringsheim 5, TTWARI, S. C, WICK, S. M., WILLIAMSON, E. & GUNNING, B. E. S. (1984). Cytoskeleton and integration of cellular function in cells of higher plants. J. Cell Biol. 99, 63s-69s. WARDROP, A. B. (1983). Evidence for the possible presence of a microtrabecular lattice in plant cells. Protoplasma 115, WICK, S. M. & DUNIEC, J. (1983). Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. I. Pre-prophase band development and concomitant appearance of nuclear envelope-associated tubulin. J. Cell Biol. 97, WICK, S. M. & DUNIEC, J. (1984). Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. II. Transition between the pre-prophase band and the mitotic spindle. Protoplasma 122, {Received 4 June Accepted 14 August 1985)