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 micro- tubules 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 com- pleted 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 micro- fibrils 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 schizo- genous 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, trans- verse 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 ob- served 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 micro- tubules 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