Microtubules and Guard-Cell Morphogenesis in Zea Mays L

J. Cell Set. 45, 211-244 (1980) 211 Printed in Great Britain © Company of Biologists Limited 1080

MICROTUBULES AND GUARD-CELL MORPHOGENESIS IN ZEA MAYS L.

B. GALATIS Institute of General Botany, University of Athens, Athens 621, Greece

SUMMARY In median paradermal planes of very young guard cells of Zca mays, numerous anticlinally oriented microtubules line densely the whole length of the ventral wall. In the external and internal regions of this wall, the subplasmalemmal microtubules are restricted to the middle of its length, where local thickenings start being deposited. In periclinal walls they were observed converging from their ends towards the thickenings. Few microtubules are present in the rest of the anticlinal walls. Before initiation of the thickenings, the parietal cytoplasm of the periclinal walls around the middle of the ventral wall contains a large number of microtubules diverging from this region and intimately associated with numerous dictyosome vesicles. Microtubule-organizing centres (MTOCs) of both periclinal and ventral walls seem to operate in these areas. The thickening of the middle of the ventral wall is initially limited at its external and internal ends. In these regions, local pads of thickening are gradually deposited, also covering a significant part of the periclinal walls, particularly the external ones, while the microtubules around them proliferate. A number of microtubules are found at a significant distance from the thickenings. The mid-depth region of the ventral wall is obviously thickened before stomatal pore opening. In this region the microtubules also become confined around the thickening. Progressively, material is apposed to the unthickened mid-regions of the periclinal walls. In the latter, both microtubules and microfibrils exhibit a clear radial arrangement around the margins of the ventral wall thickenings. The cytoplasm surrounding them possesses abundant smooth dictyosome vesicles, exhibiting intimate associations with microtubules, and some coated ones, both positive to the PA-TCH-SP reaction. The above wall differentiation is followed by stomatal pore opening, which commences initially from the internal and later from the external ventral wall thickenings and proceeds inwards; this process is also completed in dark-grown leaves. During later stages of morphogenesis, the guard cells increase considerably in length, becoming thin in their middle region. The stomatal pore elongates, and the central canal is formed. The mid-canal microtubules do not initially exhibit a definite orientation; later they become parallel to the cell axis, an alignment followed by the microfibrils of the wall. The microtubules of the margins of the extending canal are more grouped and retain a rather consistent orientation. In the ventral wall they are usually oriented transversely to the leaf surface. In periclinal walls some of them are directed at an angle to the lateral walls bordering the terminal canal thickenings, and others radiate towards the bulbous ends of the guard cells. In this case, the microtubules also appear parallel to the microfibrils. An increased protoplasmic activity, especially marked by the proliferation of dictyosome and ER membranes, accompanies an intense thickening, initially of the periclinal and later of the ventral wall of the canal. The microtubules underlie the thickening ventral and periclinal walls, but are absent from the non-thickening lateral walls of the canal. Finally, the periclinal, the transverse, and a major part of the lateral walls of the bulbous ends of the guard cells become thickened. Microtubules again line these thickening wall regions. The observations suggest that microtubules play a critical role in guard-cell morphogenesis in Zea mays. Apart from that, extensive cell elongation seems to be an essential shaping factor of the dumbell-shaped guard cells. 212 B. Galatis

INTRODUCTION In the periclinal walls of kidney-shaped guard cells, a prominent system of microfibrils radiates from the rims of the stomatal pore towards the dorsal walls (Ziegen- speck, 1938/1939, 1955a, b; Volz, 1952). In Pisum sativum (Singh & Srivastava, 1973), Allium cepa (Palevitz & Hepler, 1976), and Vigna sinensis (Galatis & Mitrakos, 1980), the radial microfibrils are deposited close to a cytoplasmic region traversed by a microtubular system of the same orientation. In the latter plant, the schizogenous formation of the stomatal pore seems to be the immediate consequence of the shaping of the guard cell, which follows the deposition of the radial microfibrils. In Vigna sinensis guard cells, although some questions have remained unanswered, the gross pattern of the microtubule distribution seems to be related to the pattern of the wall thickening. The above information led to the conclusion that the microtubules may be the organelles underlying the morphogenesis of the kidney-shaped guard cells. It is known that in dumbell-shaped guard cells the determination of form involves not only oriented microfibril deposition (Ziegenspeck, 1938/1939), but also the formation of precisely patterned thickenings (Flint & Moreland, 1946; Brown & Johnson, 1962; Srivastava & Singh, 1972). In contrast to the kidney-shaped guard cells, the participation of the protoplast and particularly the role of the microtubules in the morphogenesis of the dumbell-shaped ones has not been investigated. Re- garding the microtubules, it is known only that in differentiating guard cells they are localized in the middle of the ventral wall, where the thickening of the future pore is formed (Pickett-Heaps, 1967; Kaufman, Petering, Yocum & Baic, 1970; Srivas- tava & Singh, 1972; Ziegler, Shmueli & Lange, 1974). A detailed description of the pattern of the wall thickening in the guard cells of Zea mays has been reported by Srivastava & Singh (1972; see also Brown & Johnson, 1962). In the present paper, the distribution and orientation of the cortical microtubules, during guard-cell differentiation in Zea mays, were examined in order to find out whether they are involved in guard-cell morphogenesis. The following p oints were particularly studied, (a) Microtubule distribution and its relationship to the pattern of wall thickening, (b) The consistency of the mutual alignment of microtubules and cellulose microfibrils, and whether the deposition of the microfibrils diverging from the margins of the canals towards the bulbous ends of the guard cells (Ziegenspeck, 1938/1939) occurs over a similarly organized microtubular system, (c) The mechanism of the schizogenous opening of the stomatal pore and whether, as in the kidney- shaped guard cells of Vigna sinensis (Galatis & Mitrakos, 1980), the organization of a radial microfibrillar system in the periclinal walls precedes this process. Morphogenesis in kidney-shaped and dumbell-shaped guard cells is compared and discussed. Microtubules and guard-cell morphogenesis 213

MATERIALS AND METHODS Two- to sixteen-day-old primary leaves of Zea mays seedlings, grown under dark or light conditions, were fixed in 3 % phosphate-buffered glutaraldehyde, containing traces of CaClt, at pH 7, at room temperature for 2 h. After a postfixation in 1 % osmium tetroxide at 4 °C for 6 h, the specimens were dehydrated in acetone, and infiltrated and embedded in Durcupan ACM (Fluka). Thin sections, double-stained with uranyl acetate and lead citrate (Reynolds, 1963), were examined with a Hitachi HS-8 or a Philips 300 electron microscope. The periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of the insoluble polysaccharides (Thi6ry, 1967) was performed on double-fixed material (for details of the method see Galatis, Apostolakos & Katsaros, 1978).

Abbreviations used in figures cdv, coated dictyosome vesicle; d, dictyosome; dv, dictyosome vesicle; eptc, external periclinal wall; er, endoplasmic reticulum; g, ventral wall gap; ipto, internal periclinal wall; ho, lateral wall; m, mitochondrion; ml, middle lamella; mt, microtubule; n, nucleus; p, plastid; pd, plasmodesma; pw, periclinal wall; sp, stomatal pore; tct, terminal canal thickening; tw, transverse wall; v, vacuole; vw, ventral wall.

OBSERVATIONS Early stages of guard-cell morphogenesis The young guard cells contain a large nucleus occupying a significant part of the cell volume and a cytoplasm densely filled with ribosomes. They are devoid of or possess only small vacuoles. Variously shaped proplastids, typical mitochondria, rough ER membranes, undeveloped microbodies, and active dictyosomes are observed in the cytoplasm (Figs. 1, 7, 8). The guard cells communicate with each other via conspicuous ventral wall gaps and with their neighbouring cells through plasmodesmata (Figs. 1,7; see also Srivastava & Singh, 1972). They gradually start growing and elongating (Fig. 7). Along their whole length, the young guard cells lie at the same level as the subsidiary and typical epidermal cells (Fig. 8). In median paradermal planes of very young guard cells, 2 groups, each of more than 50 densely arranged microtubules, line the cytoplasmic faces of the plasmalemma of the ventral wall, more or less along its whole length (Figs. 2 A-c, 30 1 D). These microtubules are anticlinally oriented, arranged 1-3 units deep into the cytoplasm and frequently linked by cross-bridges with the plasmalemma (Fig. 2A-C). At this level, the ventral wall shows almost the same thickness along its entire length. In contrast, many fewer microtubules were detected in close vicinity to the other anticlinal walls (Figs. 3, 30 1 D). At the same stage of differentiation, in the external and internal regions of the ventral wall, they are restricted to a smaller part of its middle portion, bordering a nascent wall thickening (Figs. 4, 5, 30 1 B). The cytoplasm beneath the non-thickening parts of the ventral wall at the above level is traversed by some microtubules running parallel to the leaf surface (Fig. 4). Microtubules detected in the cortical cytoplasm of the periclinal walls of very young guard cells also show a patterned organization. In paradermal sections, passing near the periclinal wall, just before the initiation of the ventral wall thickening, they are observed converging from the ends of the periclinal walls (joined with the lateral 214 B. Galatis

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: • fy Microtubules and guard-cell morphogenesis 215 and transverse ones) towards their middle region (Figs. 9, 12; see also Fig. 17). At the same time, a large number of microtubules intimately associated with numerous vesicles, are localized in the parietal cytoplasm of the periclinal walls surrounding the middle of the ventral wall (Figs. 9, 15, 16). Although these microtubules follow different orientations, they sometimes appear to fan out from this region towards the other walls (Figs. 9, 12; see also Fig. 30 ;B). Closer examination of this area reveals that groups of microtubules are focused on certain cytoplasmic areas, which sometimes appear electron-dense and contain vesicles (Fig. 9). The structure of these areas is poorly resolved. In the region of the future thickening, the subplasmalemmal microtubules are randomly oriented (compare Fig. 9 with Fig. 4). The vesicles accumulated around the stabilizing thickening give a positive reaction to the PA- TCH-SP test (Fig. n); this suggests that they carry polysaccharide substances and that they are of dictyosomal origin. The above findings favour the hypothesis that the major organizing centres of the microtubules of both the periclinal and ventral walls may function in the cytoplasm and/or on the plasmalemma around the internal and external ends of the middle of the ventral wall. In these regions the microtubule- organizing centres (MTOCs) seem to continue functioning after the initiation of the thickening; microtubules were observed converging to some cytoplasmic areas containing vesicles, in the immediate vicinity of the thickenings (Figs. 4, 10, 10 inset). The above MTOCs seem to become activated before the initiation of the ventral wall thickenings. In postcytokinetic guard cells, MTOCs with a limited activity may operate at the edges formed by the junction of the periclinal with the lateral and transverse walls. In very young guard cells there is some evidence that the microtubules diverge from the external and internal ends of the middle of the ventral wall towards its mid-depth region. In serial paradermal sections close to the above areas, the microtubules of the terminal portions of the ventral wall appear fewer and are sectioned obliquely and finally longitudinally (Fig. 30 1 B, 1 c, 1 D). In contrast, the microtubules of the mid-depth region of the ventral wall consistently exhibit an anticlinal orientation (compare Fig. 2A-C with Fig. 4). Examination of serial transverse sections of young stomata confirmed that, although the microtubules run along the whole depth of the ventral wall, a thickening starts being deposited only at the external and internal ends of its middle region (Figs. 13, 15, 16, 30 1 A; see also Fig. 10). The initiation of the ventral wall thickening is an event more or less contemporary with the development of organized arrays of micro-

Fig. 1. Very young guard cells in a median paradermal section. Note the large size of their nuclei, and the discontinuity of the ventral wall at its end (arrow), x 9500. Fig. 2A-C. Successive regions of the ventral wall illustrated in Fig. 1, at a higher magnification. Microtubules line densely almost its whole length. Many of them are linked by bridges to the plasmalemma (arrows). At this median paradermal level, the ventral wall exhibits almost the same thickness along its entire length, x 45 000. Fig. 3. The lateral wall of the guard cell, indicated by the brackets in Fig. i, at a higher magnification. Many fewer microtubules (arrows) run along its subplasmalemmal surface than along the ventral wall of the same guard cell, shown in Fig. 2A-C. x 45000. 216 B. Galatis Microtubules and guard- cell morphogenesis 217 tubules. At the stage of the beginning of the thickenings, fusion of smooth and some coated vesicles with the plasmalemma bounding them were observed (Figs. 4, 14, 15). During the succeeding stages of differentiation, the cell elongates, while the number and the distribution of microtubules are continuously changing. In median paradernial planes, they disappear from the ends of the ventral wall and before the opening of the stomatal pore they are localized in an area occupying half to one fifth of its length, bordering a thickening in process of deposition (Figs. 6, 7, 30 20, JD). Few microtubules oriented in parallel to the leaf surface may be detected in the unthickened regions of the ventral wall. At this stage, in the internal and external regions of the ventral wall thickenings, the subplasmalemmal microtubules increase in number and sometimes become more numerous than the ones lining its mid-depth region. The microtubules of the lateral walls remain fewer than the ones of the ventral wall (Fig. 30 2D). The external and internal ventral wall thickenings become gradually very conspicuous and occupy a significant part of the periclinal wall, particularly the external one; they appear lens-shaped in paradermal sections and triangular in transverse ones (Figs. 19, 30 2A, 2B). Numerous vesicles exhibiting intimate associations with microtubules accumulate around the actively growing thickenings (Figs. 4, 10; see also Figs. 6, 24, 26). It must be noted that even at advanced stages of guard-cell growth, the mid-depth region of the ventral wall remains comparatively very thin (Figs. 19, 30 2 A). It exceeds the thickness of the lateral walls before the opening of the stomatal pore (Fig. 7). In the thickened mid-depth region of the ventral wall, the microfibrils are anticlinally oriented (Fig. 25). Progressively, material is also deposited in the unthickened mid-regions of the periclinal walls. This is fairly obvious in the opening stomata (Figs. 23, 30 JA). Images suggesting a fusion of dictyosome vesicles with the plasmalemma were commonly observed. Although these fusions were observed in all regions of the anticlinal and periclinal walls, they appeared more numerous in

Fig. 4. Part of a ventral wall in a paradermal section close to the external periclinal wall (see also Fig. 5). The guard cells from, which it has been taken are at the same developmental stage as that of Fig. i. At this level, the subplasmalemmal microtubules are localized around a developing thickening. Some microtubules oriented parallel to the leaf surface are observed along the subplasmalemmal cytoplasm of the non-thickened portion of the wall. Dictyosome vesicles are detected around the thickening, particularly at its right side, at a region in which microtubules appear focused. The arrows point to coated vesicles fusing with the plasmalemma. x 40000. Fig. 5. The stoma, the ventral wall of which is shown in Fig. 4. x 3200. Fig. 6. Median paradermal section passing through a ventral wall. A distinct thickening has been deposited along its mid-length region. Comparison with Fig. 2 A—c reveals that a marked rearrangement of microtubules has occurred; they are restricted around the thickening. The arrows indicate dictyosome vesicles, x 35000. Fig. 7. Median paradermal view of the stoma, the ventral wall of which is depicted in Fig. 6 (see black frame), before the opening of the stomatal pore. Comparison with Fig. 1 shows that it has elongated, x 6000. Fig. 8. Transverse section of a young guard cell, parallel to its long axis. At this stage of differentiation, the guard cells lie on the same level as the neighbouring epidermal cells, x 5500. 15 CEL 45 218 B. Galatis

» " vr '• Microtubules and guard-cell morphogenesis 219 their thickening regions. The coated vesicles are abundant in differentiating guard cells of Zea mays (see also later stages), and, like the smooth ones, are impregnated with Ag grains after application of the PA-TCH-SP reaction (Figs. 28, 28 inset). The thickenings also gave a strong reaction (Fig. 28). Apart from the microtubules which line the ventral wall thickenings, numerous others traverse the neighbouring cytoplasm in different directions at a significant distance from the wall (Figs. 24, 30 2 A, 3 A). The formation of this microtubular system, and the general increase of the number of microtubules bordering the ventral wall thickening, favour the hypothesis that MTOCs seem to function in the cytoplasm or at the plasmalemma surrounding the above thickenings, at later stages of their growth. Microtubules radiating from some cytoplasmic sites abutting on the external and internal thickened regions of the ventral wall and on the periclinal ones were also observed in the opening stomata (Fig. 26).

Opening of the stomatal pore The examination of guard cells at about the time of the initiation of the stomatal pore opening revealed that the microtubules in the periclinal walls acquired a typical radial distribution. They fanned out from the margins of the thickenings towards the lateral and transverse walls (Figs. 21, 30 2 B). It is worth noting that the orientation of the depositing microfibrils anticipates that of the microtubules (Figs. 21, 30 2 B). However, a typical radial arrangement of microfibrils over the whole area of the periclinal wall cannot be visualized in a single section in Zea mays guard cells, as it can in kidney-shaped ones, since the periclinal walls are concave. In serial sections it was observed that in the centre of the periclinal walls both microtubules and microfibrils are directed almost perpendicularly to the lateral walls. Lateral to this region, they are oriented at an angle to the guard-cell axis; this angle decreases as we approach the terminal regions of the lateral and transverse walls (Fig. 18; compare with Fig.

Fig. 9. Section passing through the parietal cytoplasm of the middle of a periclinal wall of guard cells just before the initiation of the ventral wall thickening (compare with Fig. 4). Numerous microtubules, intimately associated with abundant dictyosome vesicles, are detected in the cytoplasm around the middle of the ventral wall. Although they follow different orientations, they appear to radiate towards the lateral wall; some of them are focused on particular cytoplasmic regions (large arrows), which may represent MTOCs. The small arrows indicate coated vesicles fusing with the plasmalemma of the ventral wall, x 50000. Fig. 10. Growing internal thickening of the ventral wall. The cytoplasm occupying the junctions of the ventral and periclinal walls is traversed by microtubules showing different orientations and displaying associations with dictyosome vesicles. MTOCs may continue to operate in these regions, x 45 000. The inset shows microtubules converging on an electron-dense cytoplasmic site. This figure has been taken from another section of the region shown by the large arrow in Fig. 10. x 65 000. Fig. 11. This figure shows the parietal cytoplasm of the middle of the periclinal wall, at a stage similar to that shown in Fig. 9, as it appears after the PA-TCH-SP test. The vesicles have positively reacted, an observation suggesting that they are of dictyosomal origin, x 40000. 15-2 220 B. Galatis The opening of the stomatal pore commences initially from the internal and later from the external thickening of the ventral wall, and proceeds inwards (Fig. 23). The forces which bring about the schizogenous ventral wall splitting must be generated by the elongation of the cell and/or the increase in amount of protoplasm and/or

Fig. 12. Diagram to show the microtubule organization in the cortical cytoplasm of the periclinal walls just before the initiation of the ventral wall thickening. It is clear that the microtubules converge towards the middle of the ventral wall. In this area, numerous dictyosome vesicles appear accumulated. The microtubules and the dictyosome vesicles in this diagram have been drawn from a section of the guard cells, parts of which are shown in Fig. 9, and a neighbouring section. Dots indicate transversely sectioned microtubules, lines denote longitudinally sectioned microtubules, and open circles are dictyosome vesicles. vacuolation. The electron-transparent appearance of the middle lamella of the ventral wall in the plant examined here was not so prominent as in Pisum sativum (Singh & Srivastava, 1973) and Vigna sinensis (Galatis & Mitrakos, 1980). A slight structural differentiation of the middle lamella was observed from the first stages of the ventral Microtubules arid guard-cell morphogenesis 221 wall thickening (Fig. 14). It becomes more obvious before the opening of the stomatal pore (Fig. 19). The middle lamella was not stained by the PA-TCH-SP test, even at the first stages of ventral wall development (Fig. 29). In contrast to Vigna sinensis, the stomatal pore in Zea mays is completed in dark-grown plants. Simultaneously with the cell shaping, a part of the middle region of the lateral walls separates from the wall bounding the subsidiary cells (Fig. 27).

Advanced stages of guard-cell morphogenesis The guard cells of a recently opened stoma of Zea mays sometimes resemble the kidney-shaped ones closely in form, in both paradermal and transverse sections (Fig. 20). At this stage, the microtubules associated with the ventral wall are restricted to its portion limiting the stomatal pore, and exhibit a more typical radial orientation around its rims (Figs. 22, 30 4 c). After the opening of the stomatal pore, the pattern of morphogenesis shifts from the one forming a more or less kidney-shaped guard cell to that determining a dumbell-shaped one, while important cytological events take place. The guard cell elongates significantly, becoming thin at its middle region, the length of the stomatal pore increases and the canal is formed (Figs. 31, 43 c). The elongation is favoured in the mid-region of the guard cell, where the microfibrils of the periclinal walls are aligned transversely or at a small angle to the lateral walls. On the other hand, the terminal regions of the guard cells, where the microfibrils are almost parallel to the cell axis, do not extend detectably. Concomitantly, the median regions of the guard cells continue to lower in relation to the epidermal surface. Guard-cell elongation is initially accompanied by a remarkable reduction in wall thickness, although compensating wall deposition takes place. The ventral and periclinal wall thickenings become less obvious, and sometimes ventral, periclinal and lateral walls do not exhibit significant differences in thickness. The outstanding cytological change in the guard cells at this stage of differentiation is the reorganization of microtubules. The microtubule population of the canal, especially in the periclinal walls, seems to be increased, possibly by the interposition of new microtubules (Fig. 32). In the periclinal walls, they are more numerous than the ones of the ventral wall, which also clearly exceed in number those of the lateral walls. In the middle of the ventral and periclinal walls of the canal, they appear traversing the cytoplasm in more than one direction (Figs. 32, 43 B, C). On the other hand, the microtubules of the terminal canal portions retain a more definite orientation; they are more grouped, parallel to one another, and in periclinal walls they are aligned at an angle to the cell axis, while in the ventral wall they run transversely to the leaf surface (Figs. 32, 43A, B, c, D). This microtubule orientation remains more or less consistent up to the completion of cell differentiation, while the microtubules of the middle of the canal tend to become parallel to the cell axis (Figs. 40, 431, j, L, M). In stomata of etiolated leaves, the radial arrangement of both microtubules and microfibrils is retained for a longer period of time after the opening of the stomatal pore (Fig. 39). This can be attributed to the smaller cell elongation (Fig. 38; compare with Fig- 36). Microtubules were also seen in both lateral and transverse walls of the bulbous 222 Microtubules and guard-cell morphogenesis 223 ends. Most of them are periclinally oriented, except for a few in transverse walls, which follow an anticlinal orientation (Fig. 43A-C). Examination of the periclinal walls of the bulbous ends of the guard cells showed that microtubules line their cortical cytoplasm almost parallel to one another and parallel or at a small angle to the cell axis. This distribution remains unaltered up to the termination of guard-cell differentiation. The microtubules appear focused on the margins of the periclinal wall of the canal, and are always parallel to the wall micronbrils (Figs. 37, 43 F, G). The periclinal walls of the canal gradually become surprisingly thick, occluding a significant part of the cell space (Fig. 34). The terminal canal thickenings are very conspicuous in both surface and transverse views, and consist of microfibrils running in the wall parallel to one another (Figs. 33, 37, 43 L). In the course of the deposition of these thickenings the immediate neighbouring cytoplasm is lined by a distinct set of microtubules (Figs. 33, 43 L; for a detailed description of the thickenings of the guard cells of Zea mays see Srivastava & Singh, 1972). During wall differentiation, dictyosomes appear particularly active and produce a large population of vesicles among which coated ones are fairly numerous (Fig. 32). The ER membranes appear to proliferate and, particularly in the canal, they form an outstanding membranous system of the cortical cytoplasm. In the canal, some of them are positioned close to the plasmalemma (Fig. 40). Although the ventral wall of the canal, at advanced stages of differentiation, is lined by more microtubules than the lateral walls, its thickness does not differ significantly from that of the lateral walls. It increases only at the final stages of guard-cell differentiation (Figs. 34, 43 M). During these stages, microtubules are detected in the immediate vicinity of the periclinal, and particularly of the ventral walls, but not of the lateral walls of the mid-canal region (Fig. 35). Their absence from the lateral walls of the canal may be correlated with the absence of a distinct thickening in them (Figs. 34, 43 M). Furthermore, the ventral wall of the bulbous ends remains relatively thinner compared to the other walls (Fig. 36). Only scattered microtubules are found adjacent to this wall. Examination of paradermal sections shows that some microtubules, aligned mainly vertically to the leaf surface, are located near the transverse walls of the guard cells, at the final stages of their thickening (Fig. 43 j). Ultimately, the transverse, the periclinal and a part of the lateral walls of the bulbous ends of the guard cells become detectably thickened but to a much lesser extent than the periclinal walls of the canal (Figs. 36, 43 j, K).

Fig. 13. Median, transverse section of a young stoma at the time of initiation of the ventral wall thickening. This process commences from the external and internal ends of its middle region (arrows), x 9500. Fig. 14. Portion of an initiating ventral wall thickening in a paradermal view. Vesicles, probably of dictyosomal origin, seem to fuse with the plasmalemma (arrows). The middle lamella exhibits a slight electron transparency, x 60000. Figs. 15, 16. The external and internal thickening regions of the ventral wall of the stoma illustrated in Fig. 13. Note the accumulation of dictyosome vesicles, the microtubules lying at some distance from the plasmalemma and the coated vesicles fusing with it in Fig. 15 (arrows); compare with Fig. 9. x 40000. B. Galatis

18 20 Microtubules and guard-cell morphogenesis 225 In an exceptional instance the lateral wall of the canal of a differentiating guard cell was intensely thickened, more than the ventral wall (Fig. 41). Microtubules flanked the lateral wall as well as the rest of the protoplast surface (Fig. 42). Other outstanding protoplasmic changes accompanying guard-cell differentiation, already described by Srivastava & Singh (1972; see also Brown & Johnson, 1962), are as follows, (a) The nucleus assumes a dumbell-like shape, and traverses the lumen of the canal (Figs. 31, 36). (b) The plastids increase in size, become filled with starch grains and form simple grana (Fig. 36). (c) The mitochondria acquire an extensive internal membrane organization, while an extensive vacuolar system does not develop. During the thickening of the wall, and the mutual rearrangement of walls between guard and subsidiary cells occurring during cell elongation, the plasmodesmata appear to be destroyed. Finally, it must be noted that guard-cell differentiation in Zea mays is a prolonged process (a fact particularly true for the wall) and that the differentiated guard cells undergo only a partial and selective protoplasmic degrada- tion, keeping a significant amount of their cytoplasm and numerous mitochondria.

DISCUSSION Microtubules and the pattern of cell wall thickening The thickening of the guard-cell wall in Zea mays is an accurately controlled process. Although wall deposition seems to occur over the whole protoplast surface, distinct thickenings arise in definite wall regions. The positional relationships between the developing local thickenings and the underlying microtubules suggest that the latter are involved in the patterning of the guard-cell wall thickening. The pattern of this thickening and the relative microtubule disposition may be summarized as follows: (1) Before the formation of the stomatal pore, the middle region of the ventral and periclinal walls is locally thickened. During this process, microtubules are restricted to the middle of the ventral wall along its whole depth, and particularly concentrated along the periclinal walls around the thickenings. (2) During the elongation which follows the opening of the stomatal pore, these thickenings weaken, and new ones, more intense, occur in the periclinal walls of the canal. At this stage, the

Fig. 17. Terminal portion of the periclinal wall of a very young guard cell, including part of its parietal cytoplasm. The microtubules converge from the direction of the lateral and transverse walls towards the middle of the ventral wall, x 30coo. Fig. 18. Section through the periclinal wall of a guard cell, at a later stage of differentiation than the guard cell shown in Fig. 17. The orientation of the recently deposited microfibrils corresponds to that of the microtubules in Fig. 17. x 40000. Fig. 19. Median transverse section of a stoma, before the opening of the stomatal pore. On the internal and external ends of the ventral wall, large pads have been deposited, which in the case of the external one cover the periclinal walls almost totally. Note that the mid-depth region of the ventral wall has been only slightly thickened (compared with the lateral ones). Note the electron transparency of the middle lamella of the thickening region, x 8000. Fig. 20. A recently opened stoma. The guard cells resemble kidney-shaped ones in form, x 9000. 226 B. Galatis Microtubules and guard-cell morphogenesis 227 majority of the guard-cell microtubule population is located in the canal and particularly in the cytoplasm adjoining the ventral and periclinal walls. The subplasmalemmal cytoplasm of the terminal canal thickenings is traversed by distinct microtubule groups. (3) Finally, the ventral wall of the mid-canal region, as well as the periclinal, the transverse, and a part of the lateral walls of the bulbous ends of the guard cells become detectably thickened. Microtubules underlie these regions. On the other hand, they are absent from the lateral walls of the canal and scarce in the ventral wall of the bulbous ends, which remains relatively thin. A survey of the literature shows that microtubules always become concentrated over developing local thickenings, and are absent from the non-thickening wall regions in: xylem elements (Hepler & Newcomb, 1964; Wooding & Northcote, 1964), endothecial cells of Lilium (Heslop-Harrison, 1968), hyalocytes of Sphagnum (Schnepf, 1973), hair cells of Cobaea (Schnepf, 1974), and peristome cells of Rhacopilum tomentosum sporangia (Schnepf, Stein & Deichgraber, 1978). The microtubules of the preprophase band in the guard-cell mother cells of Vigna sinensis seem to be involved in the formation of local wall thickenings (Galatis & Mitrakos, 1979). Despite the above, the relation between microtubule distribution and the pattern of guard-cell wall thickening in Zea mays needs further consideration. Although in young guard cells the microtubules line the whole depth of the middle region of the ventral wall, most of the wall material is localized at its external and internal ends. The mid-depth region of the ventral wall is slightly thickened at later stages. The same mode of wall differentiation functions in the canal. Although variable in number, microtubules run alongside both periclinal and ventral walls of the canal, but an intense thickening is apposed to the former; the latter becomes thickened at the completion of canal differentiation. These observations pose a question about the extent of the involvement of microtubules in the formation of local thickenings in the guard cells of Zea mays, as well as about their function. Considering, on theoretical grounds, the possible cytoplasmic contribution(s) to the emergence of local thickenings of the guard-cell wall, it seems reasonable to suggest : (a) the existence of a preferential transport of matrix and/or cellulose precursors to the rising thickenings, and (b) the local accumulation, or movement to the plasmalemma bounding the thickenings, of an increased population of cellulose-synthesizing enzyme particles and/or increased activation of the existing ones. About the wall matrix, there is evidence that it is synthesized in dictyosomes and is transported to the protoplast surface by dictyosome vesicles (Northcote, 1971a, b, 1972; Whaley, 1975). On the other hand, it is now believed, at least in higher plants, that cellulose precursors are not transported via dictyosome vesicles (Ray, Eisinger & Robinson,

Fig. 21. Paradermal section through the internal ventral wall thickening at the stage of the initiation of the stomatal pore opening. The microtubules radiate out from this region; the microfibrils show the same orientation, x 30000. Fig. 22. Section of an opened stoma through the cortical cytoplasm of the periclinal wall. A typical radial organization of microtubules around the rim of the stomatal pore can be observed (compare with Fig. 21). x 27000. 228 B. Gahtis Microtubules and guard-cell morphogenesis 229 1976); the low-molecular weight precursors of cellulose may pass directly through the plasmalemma (Northcote, 1972). In guard cells of Zea mays, the only observed activi- ties, which could be considered to manifest a preferential cytoplasmic contribution in the development of the local thickenings, are the concentration of microtubules over them and the accumulation of dictyosome vesicles with which they are positionally associated. However, there is much controversy about the real function of microtubules in the formation of the local wall thickenings as well as the operation of a directed exocytosis of dictyosome vesicles over them. Regarding the function of microtubules, it has been suggested that: (a) they are involved in the directed movement of dictyosome vesicles to the growing thickenings (Pickett-Heaps, 1966; 1968; Pickett-Heaps & Northcote, 1966; Robards, 1968; Maitra & De, 1971; Northcote, 19716; Hepler, Rice & Terranova, 1972; Brower & Hepler, 1976); (b) they play a cytoskeletal role, i.e. via cross-bridges they lift the plasmalemma against turgor pressure, forming exocytoplasmic spaces in which matrix material is accumulated (Schnepf, 1973, 1974); and (c) they function as barriers to dictyosome vesicle fusion, so that the wall material is transported to the areas of wall which intervene between the thickenings and are free of microtubules (Goosen-de Roo, 1973 a, b). The application of colchicine causes aberrant wall thickenings, a fact already attributed to the breakdown of the mechanism of directed exocytosis of dictyosome vesicles over the developing local thickenings (Pickett- Heaps, 1967; Hepler & Fosket, 1971). As an alternative view, malformed thickenings may result from the disruption of a stabilization system of wall synthesis, situated at the plasmalemma (Gunning & Steer, 1976). In this manner, microtubules may also affect the emergence of local wall thickenings. The observations presented here favour the existence of a preferential exocytosis of dictyosome vesicles over the thickenings in differentiating guard cells of Zea mays. The hypothesis that the microtubules act as a barrier to dictyosome vesicle fusion (Goosen-de Roo, 1973a, b) does not seem justified, because: (a) dictyosome vesicles fusing with the plasmalemma delimiting the thickenings have been repeatedly seen; it is not implied that dictyosome vesicles do not fuse in other wall regions, but the

Fig. 23. An opening stoma. Note the obvious thickening of the mid-depth region of the ventral wall compared with the one in Fig. 19. x 8500. Fig. 24. Higher magnification of the portion of the guard cell enclosed by the brackets in Fig. 23, in another section. The cortical cytoplasm is traversed by microtubules exhibiting associations with dictyosome vesicles. Numerous other microtubules pass through the cytoplasm far from the walls, x 30000. Fig. 25. The splitting ventral wall of the stoma shown in Fig. 23. The microfibrils as well as the microtubules (arrows) are anticlinally oriented, x 30000. Fig. 26. Higher magnification of a part of a periclinal wall and its neighbouring cytoplasm taken from another section of the opening stoma shown in Fig. 23. Micro- tubules converge to a particular cytoplasmic region (arrows), containing dictyosome vesicles, x 45 000 Fig. 27. Section through the internal periclinal wall of an opened stoma. During stomatal pore opening, a part of each lateral wall pulls apart from its partner bounding the subsidiary cell (arrows), x 9500. 23° B. Galatis i

i ••

« c l-•+*••

29 Fig. 28. External ventral wall thickening, after PA-TCH-SP staining. Note the accumulation of dictyosome vesicles impregnated with Ag grains around it. x 33 000. The inset shows a coated vesicle, which has positively reacted, x 150000. Fig. 29. Young ventral wall, after the PA-TCH-SP reaction. The middle lamella has not reacted, x 45 000. Microtubules and guard-cell morphogenesis 231 phenomenon seems more intense over the thickenings, and (b) the microtubule- dictyosome vesicle associations around the thickening are so frequent and intimate, that they cannot be considered as accidental. These relationships suggest an interaction between these cytoplasmic elements. It seems that the microtubules may be involved in dictyosome vesicle distribution along the protoplast surface. In that way they may affect the pattern of the wall thickening.

1 :

Fig. 30. Diagram showing microtubule distribution and organization in the walls of differentiating guard cells up to the opening of the stomatal pore. 1A-4 A : Median transverse plane. 1B-4B: Paradermal planes close to the external periclinal wall, corresponding to plane I in Figs, i A-4 A. 1 c-4 C: Paradermal planes corresponding to plane II in Figs. 1 A-4 A. 1D-4D: Median paradermal planes (plane III in Figs. 1A-4A). Dots denote transversely sectioned microtubules; dark lines, longitudinally sectioned microtubules; white lines, wall microfibrils.

In order to explain the differential thickening of the ventral wall, it may be suggested that either the cytoplasm promotes the thickening of these regions or particular microtubular or other conditions are prevailing there. Taking into consideration the organization and distribution of the microtubules in guard-cell walls of Zea mays, and recent detailed observations of Gunning, Hardham & Hughes (1978) implying that MTOCs generating the interphase microtubule framework function in the cytoplasm occupying the edges of postcytokinetic cells, it is hypothesized that the parietal cytoplasm of the periclinal walls around the middle region of the ventral one may con- 232 B. Galatis Microtubules and guard-cell morphogenesis 233 tain MTOCs of both the above walls. During cytokinesis, the dictyosome vesicles move preferentially towards the cell plate among the phragmoplast microtubules (Hepler & Jackson, 1968; Cronshaw & Esau, 1968); it is known that the phragmoplast MTOCs lie on the level of the cell plate, where the microtubules overlap (Inoue, 1964; Allen & Bowen, 1966; Inoue & Sato, 1967; Gunning et al. 1978). If in developing ventral wall thickenings dictyosome vesicles move in the direction of microtubules, their movement towards the MTOCs as well as their fusion with the neighbouring plasmalemma may be favoured (see also Galatis & Mitrakos, 1980). This hypothesis may also help to explain the intense thickening of the periclinal walls of the canal, while the ventral one remains initially thin, and both of them are lined by microtubules. The suggestion that prominent MTOCs function in the cortical cytoplasm of the periclinal walls surrounding the middle region of the ventral one is supported by: (a) the rinding that the cortical microtubules of periclinal and possibly ventral walls are focused in these regions; (b) the observation of microtubule-vesicle complexes, similar to the ones observed by Gunning et al. (1978), in the cell edges of Azolla, and thought to represent MTOCs; and (c) that in postcytokinetic guard cells, the re-positioned microtubules appear at first in the periclinal and terminal anticlinal wall regions. Regardless of the above, the initiation and especially the orientation of cortical microtubules in guard cells of Zeamaysmust be a much more complicated phenomenon. The continuous changing of microtubule organization suggests that MTOCs of controlled capacity are sequentially activated in different regions of the cell, possibly at the cell edges, although some evidence was obtained only for prominent ones functioning in the cortical cytoplasm of the periclinal walls surrounding the middle of the ventral one. The observations presented here need further experimental and structural documentation. In this direction, the existence of MTOCs functioning during the microtubule reorganization which follows the opening of the stomatal pore, as well as the deployment of microtubules during the time between the completion of cytokinesis and the initiation of the ventral wall thickening must be investigated. During this stage, the microtubule orientation seems to be highly change- able. Recent evidence has cast some doubt on the concept that microtubules are involved in the transportation of cytoplasmic particles, on the basis that they cannot provide the force required for movement (for a review see Gunning & Steer, 1976). Moreover, in the mitotic spindle the microtubules have been detected spatially associated with actin-like fibrils; it has been considered that these fibrils co-operate with microtubules in chromosome movement, providing the necessary force (Forer & Jackson, 1976,

Fig. 31. Median paradermal section of an elongating stoma. Compare with the one of Fig. 7. Note the elongation of the stomatal pore, the shape of the nuclei, and the position of the vacuoles. x 7000. Fig. 32. Micrograph depicting a part of the periclinal wall of an expanding canal and its cortical cytoplasm. The microtubules of its mid-regions do not show a definite orientation. On the contrary, at the margins of the canal, they appear parallel to one another, and directed at an angle towards the lateral walls (arrows), x 22000. 16 CEL 45 B. Galatis Microtubules and guard-cell morphogenesis 235 1979; Forer, Jackson & Engberg, 1979). In this case, the microtubules might function only to determine the direction of transportation of cytoplasmic particles (Roberts, 1974). In cells of higher plants, the existence of a microfilamentous or other system spatially or functionally associated with subplasmalemmal microtubules has not yet been visualized. Freundlich (1974), however, reported that in elongating cells of Nym- phoides indica microtubules interact with microfilaments and suggested that the latter provide the force required to drive the dictyosome vesicles to the sites of their fusion. Microfilaments have been observed in a few types of elongated plant cells and not in the abundance in which they have been identified in animal cells (Parthasarathy & Miihlethaler, 1972). No further discussion is possible until we obtain more information. The increased microfibril synthesis in the local thickenings could be explained by the suggestion that the cellulose-synthesizing enzymes associated with the plasmalemma are accumulated and/or stabilized by microtubules. However, no relevant information for discussion has been obtained from the present study. Data bearing on such an activity can be obtained only from studies using the freeze-etching technique (for a recent review see Brown & Willison, 1977). It has also been suggested, but not yet confirmed, that by means of dictyosome vesicles, cellulose synthetases are transported to the plasmalemma of the growing wall (Northcote, 1972; Ray, Shininger & Ray, 1969; Ray et al. 1976). In conclusion, it can be said that although microtubules seem to play an indis- pensable role in the appearance of the local guard-cell wall thickenings, definite answers about their real function cannot be given. However, if in Zea mays guard cells other cytoplasmic elements or factors are implicated in this process, they must be related either to microtubules or to the regions in which MTOCs are believed to function. In guard cells of Zea mays, an ER membrane distribution connoting their direct participation in the patterning of the wall thickenings was not observed. ER portions were located close to the thickenings, as well as in other cell regions. The prominence of ER membranes in differentiating guard cells, particularly in their canals, implies that they might be directly or indirectly involved in wall thickening, a function repeatedly suggested, but not yet proven (for reviews see Roberts, 1969; Roland, 1973). Besides, the close ER membrane-plasmalemma juxtapositions in the canal may

Fig. 33. Transverse section through the margins of the canal at the time of completion of the wall thickening.The microtubules in the ventral wall appear oriented perpendicularly to the periclinal walls (double arrows). Cross-sectioned microtubules are detected over the terminal canal thickenings (single arrows), x 25 000. Fig. 34. Mid-canal region of a more or less differentiated guard cell, in transverse view. Note that compared with the lateral wall, the ventral one has increased in thickness, x 20000. Fig. 35. Higher magnification of a portion of the guard cell shown in Fig. 34 in another section. The cytoplasm abutting on the thickening ventral wall is crossed by microtubules parallel to the cell axis (arrows), while that of the lateral one is devoid of microtubules. x 45 000.

16-3 B. Galatis

m*$ a s"

36 5 Microtubules and guard-cell morphogenesis 237 also be related with the plasticization of the expanding wall. Such a hypothesis has been made for similar relationships found in polarizing mucilage papillae mother cells of Marchantia (Galatis & Apostolakos, 1977). It is known that the wall thickening of dumbell-shaped guard cells (Flint & More- land, 1946; Brown & Johnson, 1962; Srivastava & Singh, 1972) is quite different from that of kidney-shaped ones (see Guttenberg, 1959). The observations presented here show that in Zea mays, the mode of ventral wall thickening preceding the opening of the stomatal pore is quite similar to that of kidney-shaped guard cells (Stevens & Martin, 1978; Galatis & Mitrakos, 1980; see also Esau, 1965). Comparison of the guard cells of Zea mays with the kidney-shaped ones of Viciafaba (Singh & Srivastava, 1973), Allium cepa (Palevitz & Hepler, 1976), and Vigna sinensis (Galatis & Mitrakos, 1980), led to the conclusion that this similarity extends to the fine details of the thickening process. The existence of a microtubular system lining the thickening of the future pore has been confirmed in all the kidney-shaped (Landre', 1969; Singh & Srivastava, 1973; Peterson & Hambleton, 1978; Galatis & Mitrakos, 1980), and dumbell-shaped (Pickett-Heaps, 1967; Kaufman et al. 1970; Srivastava & Singh, 1972; Ziegler et al. 1974) guard cells examined. The microtubule arrangement in the young guard cells of Zea mays differs from that of the kidney-shaped ones of Vigna sinensis. In the latter plant, the microtubules of the ventral and dorsal walls do not differ significantly in number. On the contrary in Zea mays, the microtubular systems in the lateral and transverse walls are obviously subordinate compared with those of the ventral and periclinal walls. Moreover, the terminal dorsal wall thickenings of Vigna sinensis stomata deposited in guard-cell mother cells in positions marked by the microtubules of the preprophase band (Galatis & Mitrakos, 1979) were not observed in Zea mays.

Microtubules and microfibril orientation Ziegenspeck (1938/1939) discovered that in mature dumbell-shaped guard cells, the microfibril organization is quite different from that in kidney-shaped ones. In the canal, the microfibrils are oriented longitudinally to the cell axis, an observation confirmed by Setterfield (1957) with the electron microscope. Besides, they diverge from the margins of the canal towards the bulbous ends. According to Aylor, Parlange & Krikorian (1973) the latter microfibril orientation is fundamental for stomatal movement. Before the opening of the stomatal pore, the microfibril organization in the peri-

Fig. 36. A light-grown stoma, at a stage close to completion of guard-cell differentiation, x 6000. Fig. 37. Paradermal section through the margins of the periclinal wall of the canal. Microtubules (arrows) appear diverging from this region towards the ends of the guard cell. The microfibril orientation coincides with that of the microtubules. x 45000. Fig. 38. A differentiated stoma of a 7-day-old etiolated leaf. It is less elongated than the one shown in Fig. 36. x 4000. 238 B. Galatis

sT ' Microtubules and guard-cell morphogenesis 239 B c

* A \1 J | h 1 11

Fig. 43. Diagram to illustrate the microtubule organization in the guard cells, at advanced stages of their differentiation (compare with Fig. 30). A-E: Initial stages of canal formation, A-C: Paradermal planes passing through the periclinal wall of the canal (A), its cortical cytoplasm (B), and its mid-depth region (c). D, E: Transverse views, corresponding to the planes I and II of Fig. 43 c. F—M: Advanced stages of guard-cell differentiation, F, G : Paradermal planes through the periclinal wall of the bulbous ends (F), and its cortical cytoplasm (G). H, I : Para- dermal planes including the periclinal wall of the canal (H), its cortical cytoplasm, and a median region of the bulbous ends (1). j: Median paradermal plane, K-M : Transverse views, corresponding to the planes I, II and III in Fig. 43 j. Conventions as in Fig. 30.

Fig. 39. Surface section through the thickened mid-portion of an etiolated stoma at the same stage of differentiation as the one in Fig. 38. The radial microtubule arrangement is retained, although the stomatal pore has elongated, x 19000. Fig. 40. Portions of a mid-canal region of an elongated stoma, including the ventral walls. Microtubules oriented in parallel to the cell axis (arrows), and ER membranes apposed on the plasmalemma can be observed, x 35000. Fig. 41. Median transverse section of a guard cell passing through the canal. Note the unusual intense thickening of the lateral wall, x 15000. Fig. 42. Higher magnification of the protoplasm of the guard cell shown in Fig. 41. Microtubules are detected at the lateral wall as well as at the rest of the cytoplasmic surface (arrows), x 55000. 240 B. Galatis clinal walls of the guard cells of Zea mays differs considerably from the one described by Ziegenspeck (1938/1939) in mature dumbell-shaped guard cells. It was observed for the first time that the microfibrils radiate initially from the margins of the ventral wall thickenings and later from the rims of the stomatal pore towards the other anticlinal walls. The microtubule orientation matches that of the microfibrils. The radial microtubule arrangement is similar to the one described in kidney-shaped guard cells of Pisum sativum (Singh & Srivastava, 1973), Allium cepa (Palevitz & Hepler, 1976), and Vigna sinensis stomata (Galatis & Mitrakos, 1980). In the mid-depth region of the ventral wall the microfibrils are anticlinally oriented. The guard cells of a recently opened stoma of Zea mays resemble the kidney-shaped ones in form. The change of their shape into the dumbell-like one keeps pace with the change of the radial microtubule and microfibril organization. The deposition of the differential thickenings of the ventral and periclinal walls and the organization of a radial microfibrillar system in the latter, is followed by (a) the change of the guard-cell shape to the dumbell-like one, and (b) the opening of the stomatal pore. The schizogenous opening of the stomatal pore is the consequence of the beginning of guard-cell morphogenesis; it always follows the deposition of the radial microfibrils in the periclinal walls. The shift of the morphogenetic mechanism, which forms the more or less kidney- shaped young guard cells of Zea mays, into another, determining the formation of dumbell-shaped guard cells, seems to be performed by the extreme elongation of the cell. The dumbell-like shape seems to be the outcome of the interplay between the elongation and the factors underlying the oriented deposition of the wall microfibrils. Due to the radial organization of microfibrils in the periclinal walls of the young guard cells, the elongation is favoured in their middle region and disfavoured at their ends. The strains generated by the elongation are possibly responsible for the reorientation of the mid-canal microtubules which become parallel to the cell axis. An approxima- tely radial microtubule-microfibril arrangement is retained on the margins of the canal during further cell differentiation. At all stages the microtubule orientation resembles that of microfibrils. In dark-grown stomata in which cell elongation is slower than in light-grown stomata, the radial microtubular system in the periclinal walls is retained for longer. Unlike the situation in dumbell-shaped guard cells, in kidney-shaped ones the radial microtubular arrangement is preserved up to the final cell differentiation (Palevitz & Hepler, 1976; Galatis & Mitrakos, 1980). The functional significance of the coincidence of the microfibril-microtubule alignment has now been confirmed in a wide range of plant cells and it is a subject of intense investigation. A current hypothesis is that via cross-bridges, the microtubules affect the oriented microfibril deposition by the alignment of cellulose synthetases on the surface of the plasmalemma or of the microfibrils themselves (Cronshaw, 1967; Hepler & Fosket, 1971; Hepler & Palevitz, 1974; Heath, 1974). A strong supporting argument is the misalignment of the depositing cellulose microfibrils after destruction of the microtubules by colchicine (Green, 1962, 1963; Hepler & Fosket, 1971; Srivastava, Sawhney & Bonettemaker, 1977; Hogetsu & Shibaoka, 1978; Microtubules and guard-cell morphogenesis 241 Quader, Wagenbreth & Robinson, 1978; Marchant & Hines, 1979). However, counter views also exist; the colchicine may directly affect either the plasmalemma or the associated enzymes (Gunning & Steer, 1976) or a common factor may orient both microtubules and microfibrils (Green, Erickson & Richmond, 1970). The main conclusion of the present study is that the microtubules constitute a highly dynamic system in the differentiating guard cells of Zea mays; although their number, distribution and orientation are continuously changing, their overall organization remains consistent at every stage of differentiation and in relation to every particular wall. Their disposition suggests that they play a critical role in guard-cell morphogenesis, implicated in the patterning of the wall thickening and in microfibril orientation. Prominent MTOCs seem to function in the cortical cytoplasm of the middle region of the periclinal wall surrounding the ventral one, where local thickenings are deposited. The elongation of the guard cells seems to be the factor which brings about the shift of the morphogenetic pattern from the one forming kidney- shaped guard cells to that determining dumbell-shaped ones.

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