Tansley review. The role of the cytoskeleton in the morphogenesis and function of stomatal complexes. Review. Basil Galatis and Panagiotis Apostolakos

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1 Review Blackwell Publishing, Ltd. The role of the cytoskeleton in the morphogenesis and function of stomatal complexes Author for correspondence: Basil Galatis Tel: Fax: Basil Galatis and Panagiotis Apostolakos Department of Botany, Faculty of Biology, University of Athens, Athens Greece Received: 17 July 2003 Accepted: 21 October 2003 doi: /j x Contents Summary 613 I. Introduction 614 II. Cytoskeleton and development of the stomatal complexes 614 III. Cytoskeleton and stomatal cell shaping 620 IV. Stomatal pore formation 624 V. Substomatal cavity formation 625 VI. Stomatal complex morphogenesis in mutants 626 VII. Cytoskeleton dynamics in functioning stomata 628 VIII. Mechanisms of microtubule organization in stomatal cells 631 IX. Conclusions-perspectives 634 References 635 Summary Key words: cytoskeleton, morphogenesis, stomatal complexes, microtubules, actin filaments. Microtubules (MTs) and actin filaments (AFs) form highly organized arrays in stomatal cells that play key roles in the morphogenesis of stomatal complexes. The cortical MTs controlling the orientation of the depositing cellulose microfibrils (CMs) and affecting the pattern of local wall thickenings define the mechanical properties of the walls of stomatal cells, thus regulating accurately their shape. Besides, they are involved in determination of the cell division plane. Substomatal cavity and stomatal pore formation are also MT-dependent processes. Among the cortical MT arrays, the radial ones lining the periclinal walls are of particular morphogenetic importance. Putative MT organizing centers (MTOCs) function at their focal regions, at least in guard cells (GCs), or alternatively, these regions either organize or nucleate cortical MTs. AFs are involved in cell polarization preceding asymmetrical divisions, in determination of the cell division plane and final cell plate alignment and probably in transduction of stimuli implicated in stomatal complex morphogenesis. Mature kidney-shaped GCs display radial AF arrays, undergoing definite organization cycles during stomatal movement. They are involved in stomatal movement, probably by controlling plasmalemma ion-channel activities. Radial MT arrays also persist in mature GCs, but a role in stomatal function cannot yet be attributed to them. New Phytologist (2004) 161: New Phytologist (2004) 161:

2 614 Review I. Introduction The stomatal complexes are epidermal structures of the aerial organs of higher plants and very rarely of the young primary roots (Christodoulakis et al., 2002). They exhibit a structural symmetry and an integrated function, properties that are the outcome of a precise developmental sequence and a complicated differentiation. Each of them consists of two highly differentiated cells, the GCs, which border an epidermal intercellular space- the stomatal pore- and in many plants are surrounded by one or more subsidiary cells (SCs; Fig. 1a). The GC pair and the included stomatal pore are defined as a stoma. The cells, which surround a stoma and differ in size, shape, arrangement and structure from the typical epidermal cells, are defined as SCs. The stomata are capable of sensing a multitude of environmental signals to adjust properly the stomatal pore in order to regulate gas exchange in and out of the plant organs. According to Raschke (1979), the stomata function like turgor operated valves. The GCs have been equipped with a specific wall structure and a unique physiology, properties that become functionally coordinated in stomatal movement (Ketellapper, 1963; Zeiger et al., 1987; Wilmer & Fricker, 1996). Changes in turgor induce alterations in GC shape and bring about opening and closing of the stomatal pore. The GCs display two highly specialized forms: the kidneylike and the dumbbell-like one (Fig. 1b,c). The kidney-shaped GCs dominate among most plant families, while the dumbbellshaped ones are only found in Poaceae and Cyperaceae. The anticlinal wall between the GCs is termed the ventral wall (VW), while those adjacent to the surrounding cells are the dorsal or lateral ones (Fig. 1a). The periclinal walls of the GCs are designated as external and internal ones (Fig. 1a). The stomatal complexes have been the subject of intense research work by cell biologists, because they are model systems to examine the role of cytoskeleton in: cell shaping, the establishment of cellular polarity and the control of the plane of cell division in multicellular systems. Several aspects of MT and AF involvement in morphogenesis of stomatal complexes have been reviewed so far (Hepler, 1981; Palevitz, 1981a, 1982, 1991; Sack, 1987; Cleary, 2000). Besides, the stomata have proved to be an intriguing and important research object for plant physiologists (Hetherington, 2001; Schroeder et al., 2001). This article attempts an overall review of the existing information on the organization and role of MTs and AFs during morphogenesis and function of stomatal complexes. Recently, the research interest has been focused on the cytoskeleton of mature GCs since the emerging evidence reveals that cortical radial AF arrays and probably cortical radial MT arrays are involved in stomatal movement (Hwang et al., 2000). II. Cytoskeleton and development of the stomatal complexes Fig. 1 (a) Three-dimensional representation of an elliptical stoma. DW, dorsal wall; EPW, external periclinal wall; GC, guard cell; IPW, internal periclinal wall; SC, subsidiary cell; VW, ventral wall. (b, c) Diagrams of an elliptical (b) and a dumbbell-shaped (c) stoma. The dotted lines indicate the orientation of the cellulose microfibrils. BE, bulbous ends; C, canal; GC, guard cell; SC, subsidiary cell (from Meidner & Masnfield eds, Physiology of Stomata, McGraw-Hill, London, 1968). 1. Developmental processes The stomatal complexes are generated by one or more differential divisions, which are usually asymmetrical and produce the guard cell mother cell (GMC) and the SC(s), and by a symmetrical division, which yields the GCs. There are three ontogenetical types of stomatal complexes: the mesogenous in which the SCs have a common origin with the GCs, the perigenous where the SCs are derived from protodermal cells other than those cutting off the GCs, and the mesoperigenous in which the SCs are of mixed origin. In the perigenous stomatal complexes, the GMC induces asymmetrical divisions New Phytologist (2004) 161:

3 Review 615 Fig. 2 Three-dimensional representation of a leaf protodermal area of Zea mays at the stage of SC formation. EPW, external periclinal wall; GMC, guard cell mother cell; IPW, internal periclinal wall; LW, lateral wall; SC, subsidiary cell; TW, transverse wall (from Galatis, 1982). in some or all the adjacent cells (subsidiary cell mother cells; SMCs) that separate SCs (Stebbins & Jain, 1960; Stebbins & Shah, 1960, Fig. 2). In Vigna sinensis, which forms mesogenous stomata, the early formed leaf and hypocotyl stomata are large in size and lack SCs. These stomata have the ability to induce oriented divisions in their neighbouring cells, generating numerous SCs (Galatis & Mitrakos, 1979, 1980; see also Christodoulakis et al., 2002). In mesogenous stomatal complexes, the number of the SCs seems to depend on the developmental stage of the protoderm. For instance, in very young leaves of Vigna sinensis (Galatis & Mitrakos, 1979) and Arabidopsis thaliana (Zhao & Sack, 1999) the stomata lack SCs or display one SC, while in more mature leaves the number of the SCs increases. Light (Kazama & Mineyuki, 1997) and increasing CO 2 levels (Boetsch et al., 1996) induce an increase in the number of SCs in Cucumis sativus hypocotyls and Tradescantia virginiana leaves, respectively. Although the term stomatal cells strictly refers to the GCs, we use this term here in a broad sense to define all the cells involved in development of stomatal complexes. 2. Cytoskeletal markers of polarization in stomatal cells The accumulation of cytoplasm, the migration of the nucleus and the organization of a preprophase MT band (MT-PPB) at the polar end of the cell manifest structurally the establishment of polarity in the asymmetrically dividing stomatal cells. The first two phenomena are visible in large cells, while the eccentric disposition of the MT-PPB is usually the only consistent premitotic marker of the polarized region in stomatal cells dividing asymmetrically (Galatis, 1974; Galatis & Mitrakos, 1979). Well-organized MT-PPBs precede stomatal asymmetrical divisions in the plants examined so far (Gunning, 1982; Mineyuki, 1999). The PPB region has a rather complex organization. Apart from the MT-PPB, an AF-PPB forms in the GMC progenitors of Allium cepa (Mineyuki & Palevitz, 1990) and in SMCs of Tradescantia virginiana (Cleary, 1995; Cleary & Mathesius, 1996; Cleary, 2001) and Zea mays (Gallagher & Smith, 1999; Smith, 2001). In the latter two cases, the AF- PPBs are closely similar to the MT-PPBs and disorganize at the end of prophase leaving an AF-depleted zone persisting in mitosis and cytokinesis. This delimits the PPB region in a negative sense (Cleary, 2000; Smith, 2001). Both cytoskeletal structures are probably essential for the establishment of a functional PPB region. Moreover, in Zea mays SMCs the cdc 2 kinase resides in the PPB region (Colosanti et al., 1993). Although in some plant cell types the PPB position seems to be determined by the nucleus (Mineyuki et al., 1991, Mineyuki, 1999), in the asymmetrically dividing stomatal cells there is convincing evidence that it is defined by intercellular morphogenetic stimuli. This view is supported by the facts that: (a) the MT-PPB usually appears before nuclear migration to the polarized end of the SMCs of grasses (Galatis et al., 1983a, 1984b) and GMC progenitors of Allium cepa (Mineyuki & Palevitz, 1990) and (b) the displacement of the nucleus from the polarized end of the grass SMCs by centrifugation (Pickett-Heaps, 1969a; Galatis et al., 1984a) and cytochalasin B treatment (Cho & Wick, 1990) does not affect the MT-PPB assembly laterally to the GMC. However, the strongest evidence that local transcellular morphogenetic stimuli define the PPB position has been derived from the study of single SMCs of three Triticum species, being laterally in contact with two GMCs. When these SMCs are simultaneously induced to divide by the GMC pair, two MT- PPBs are assembled, one laterally to each GMC ( double polarized SMCs ; Galatis et al., 1983a; Fig. 3a). Moreover, in bi-spaced binuclear SMCs of Triticum vulgare and Zea mays produced by caffeine treatment one MT-PPB is assembled laterally to the inducing GMC (Pickett-Heaps, 1969b; Apostolakos & Galatis, 1987). Therefore, the number and the position of the MT-PPBs is not related to the number of the nuclei, but is defined by the morphogenetic stimuli functioning in the dividing stomatal cells, a phenomenon also confirmed in other plant cell types (Manandhar et al., 1996a,b). Fragmentary but important information is available on the mode of formation of the unique MT-PPBs in grass SMCs. These MT-PPBs are curved and line a limited region of the cell cortex transversely to the preceding interphase cortical MT arrays (Fig. 7b). They are formed by sets of antiparallel MTs, initiated by MT foci residing at the cell edges made by the periclinal walls with the anticlinal one facing the stomatal row, where putative MTOCs possibly function (Cho & Wick, 1989; Wick, 1991a). In colchicine-affected SMCs, the wall regions adjacent to the MT-focal sites are locally thickened in the absence of MTs, an observation revealing the polarizing nature of the underlying cortical cytoplasm (Galatis et al., 1984a). New Phytologist (2004) 161:

4 616 Review Fig. 3 (a) Diagram of a double polarized SMC of Triticum in a paradermal view.the dark lines show the position of the two MT- PPBs. (b d) The arrangements of the SC wall in the divided double polarized SMCs. GMC, guard cell mother cell; N, nucleus; SMC, subsidiary cell mother cell. In SMCs of the grasses Secale cereale (Cho & Wick, 1990, 1991) and Zea mays (Gallagher & Smith, 1999; Smith, 2001) and in the lateral SMCs of the monocotyledon Tradescantia virginiana (Cleary, 1995; Cleary & Mathesius, 1996; Cleary, 2000), apart from the MT-PPB and the AF-PPB, distinct cortical AF patches appear beneath the plasmalemma adjacent to the wall region shared with the GMC (Fig. 4a). They consist of thick AF bundles perpendicular to the epidermis. These bundles enter the cortical cytoplasm abutting the SMC periclinal walls and mark the polar region of the SMCs before mitosis. Double polarized SMCs have also been found in Tradescantia virginiana, where two AF patches are localized, one laterally to each GMC (Cleary & Mathesius, 1996). In the same plant, AFs accumulate on the transverse anticlinal wall of the GMC progenitors closest to the polarized nucleus. By contrast to the MT-PPB and the AF-PPB, the AF patches persist in mitosis and cytokinesis and are more resistant to cytochalasin B treatment than the fine endoplasmic and cortical AFs (Cho & Wick, 1990, 1991; Kennard & Cleary, 1997). In the plasmolyzed SMCs of Tradescantia virginiana the plasmalemma adjacent to the PPB region and to the cortical AF patches is not detached from the cell wall, a phenomenon implying the existence of a relatively strong connection between the cell wallplasmalemma-cytoskeleton in these regions (Cleary, 2001). The AF patches participate in the polarization mechanism of the SMCs (Cho & Wick, 1990; Cleary, 1995; Cleary & Mathesius, 1996; Cleary, 2000, 2001; Smith, 2001; see section II.3) but they are not the prime-polarizing factor of the asymmetrically dividing cells. They appear in the course of transduction of the inductive stimulus, like the MT-PPB and the AF-PPB, after migration of the nucleus. They are not so distinct in the terminal SMCs of Tradescantia virginiana (Cleary & Mathesius, 1996) and also they coexist with similar AF patches abutted on the lateral GMC walls shared with the adjacent SMCs (Cho & Wick, 1990, 1991; Cleary & Mathesius, 1996; Cleary, 2000, 2001). Fig. 4 AF organization in stomatal cells of Tradescantia virginiana. Bars, 10 µm (from Cleary & Mathesius, 1996) (a) Optical section through the mid-plane of a developing stomatal complex. The arrowheads indicate the AF patches on the anticlinal wall of the polarized lateral SMCs and the adjacent GMC. (b) Radial AF array on the periclinal face of an interphase GMC. New Phytologist (2004) 161:

5 Review 617 Fig. 5 (a, b) Migration of the nucleus in a living SMC of Tradescantia virginiana away from the adjacent GMC (asterisk) towards the site where pressure is applied by a needle (nt). Bar, 10 µm (from Kennard & Cleary, 1997). The cortical AF patches may form to protect plasmalemma regions being under mechanical stresses. In such conditions the animal cells form AF gatherings to reinforce the stressed plasma membrane regions (Ingber, 1997; Frame & Sarelius, 2000; Ko & McCulloch, 2000), a phenomenon also observed in plasmolyzed leaf cells of Chlorophyton comosum (Komis et al., 2002a). The AF patches in SMCs line the wall region shared with the lateral walls of the adjacent GMCs that elongate appreciably (Cho & Wick, 1989; Cleary & Hardham, 1989; Kennard & Cleary, 1997, see also section III.1.1 and Fig. 2). This elongation may trigger AF patch formation, a hypothesis supported by the following findings: first AF patches line both sides of the common wall between SMC and GMC, while the young GMCs lack them (Cho & Wick, 1990, 1991). Second the AF patches are retained in the young SCs of Tradescantia virginiana (see Fig. 13 in Cleary & Mathesius, 1996) and Zea mays (our unpublished data). At this stage the adjacent lateral GMC walls continue to elongate (see Figs 12 and 13 in Cleary & Mathesius, 1996). Third in plasmolyzed SMCs of Tradescantia virginiana, the AF patches extend into neighbouring cortical regions, which have been detached from the cell wall (Cleary, 2001). 3. Nuclear migration-mitotic spindle axis stabilization The correct positioning of the nucleus and the proper orientation of the spindle axis are premitotic events essential for the correct placement of the new cell wall. In the monocotyledon stomatal complexes examined so far, the nuclear position at the polar end of the GMC progenitors is controlled by transcellular morphogenetical gradients functioning along the stomatal rows, while in SMCs by stimuli emitted by the GMCs (Stebbins & Jain, 1960; Stebbins & Shah, 1960; Croxdale, 1998). In SMCs of Tradescantia virginiana, the polar nuclear migration occurs at the G 1 stage of the cell cycle (Kennard & Cleary, 1997). In the double polarized SMCs (see section II.2) the nuclear migration is controlled by stimuli emitted by both the GMCs. When they are equal in strength, the nucleus occupies a position at the mid-distance between the GMCs (Fig. 3a), but where they are unequal, it is placed close to one GMC (Galatis et al., 1983a, 1984b; Kennard & Cleary, 1997). In the latter case, the preprophase/prophase nucleus often forms an angular MT-flanked protrusion directed towards the stronger inducing GMC (Galatis et al., 1983a). It is generally accepted that nuclear migration in the asymmetrically dividing stomatal cells is controlled by AFs (Cho & Wick, 1990, 1991; Mineyuki & Palevitz, 1990; Wick, 1991b; Kennard & Cleary, 1997; Pickett-Heaps et al., 1999; Cleary, 2000; Smith, 2001), although the MT involvement has also been reported (Galatis et al., 1984a; Kazama et al., 1995). In SMCs of Secale cereale, Zea mays and Tradescantia virginiana, the SMC nucleus is stabilized by an AF patch on the lateral wall of the inducing GMC, until completion of cell division (Wick, 1991b; Cleary, 2000; Smith, 2001). Cytochalasin treatment of the above SMCs inhibits the polar movement of the nucleus (Cho & Wick, 1990, 1991; Kennard & Cleary, 1997; Gallagher & Smith, 1999; Pickett-Heaps et al., 1999). Similarly, in SMCs of Tradescantia virginiana and Zea mays interactions between endoplasmic AFs and the cortical AF patches probably anchor the one pole of the mitotic spindle close to the inducing GMC (Pickett-Heaps et al., 1999; Cleary, 2000, 2001; Smith, 2001), by a mechanism similar to that functioning in Fucus zygotes and in yeasts (Fowler & Quatrano, 1997; Drubin, 2000). Cytochalasin treatment dislodges the mitotic spindle pole from the SMC wall and the whole spindle moves away from the GMC (Pickett-Heaps et al., 1999). 4. Induction stimuli The induction of asymmetrical divisions producing the GMCs in the hypocotyl protoderm of Arabidopsis thaliana is probably a hormone-dependent process (Saibo et al., 2003). Besides, the stimulus, which is emitted by the GMCs and induces asymmetrical divisions in the grass SMCs might also be a hormone-like substance (Stebbins & Jain, 1960; Stebbins & Shah, 1960; Pickett-Heaps & Northcote, 1966). The SMCs of Tradescantia virginiana polarize at the G 1 stage of the cell cycle and remain polarized for 22 h, while the GMCs stay at the G 1. SMC polarization during G 1 stage and mitosis may be induced by different signals emitted from the GMC or some master signal dictates both processes (Kennard & Cleary, 1997). The stimulus in grass SMCs persists for a relatively long period (Pickett-Heaps, 1969b; Apostolakos & Galatis, 1987). Observations supporting the chemical nature of the stimulus have been made in incompletely divided caffeineaffected SMCs of grasses. Some of them, which display a SC wall having a minute gap, are re-induced to divide and assemble a MT-PPB at the site of the previous MT-PPB, inside the incomplete SC (Apostolakos & Galatis, 1987). In New Phytologist (2004) 161:

6 618 Review these SMCs, the nucleus(i)- and later the one mitotic spindle pole are stabilized close to the SC wall gap through which the putative inductive stimulus possibly entered the larger cell compartment (Apostolakos & Galatis, 1987). Apart from the above, the possibility that local mechanical stresses applied on the asymmetrically dividing stomatal cells by their neighbours may trigger cell polarization and/or define the PPB position should be also considered (Green et al., 1970; Galatis & Mitrakos, 1979; Pickett-Heaps et al., 1999). For instance, in grasses, the elongating GMCs (Figs 2, 7a c) may exert mechanical stresses on the adjacent SMC wall region (see section III.1.1). They may induce local differentiation in the cell wall and the adjacent plasmalemma generating asymmetries on the SMC surface, triggering mechanisms establishing polarity in it (Fowler & Quatrano, 1997). A cortical asymmetry is revealed in the plasmolyzed polarized SMCs of monocotyledons, where the protoplast remains attached on the cell wall region shared with the GMC (Stebbins & Jain, 1960; Stebbins & Shah, 1960; Cleary, 2001). The stresses could activate stretch-activated channels in the plasmalemma, causing the influx of ions, which could be involved in SMC polarization (Kennard & Cleary, 1997; Cleary, 2000). Kennard & Cleary (1997) presented convincing evidence that mechanical stresses induce polarity in SMCs. They applied pressure by a fine needle on individual SMCs of Tradescantia virginiana opposite to the adjacent GMC and Fig. 6 (a f) Diagrammatic representation of selected serial paradermal sections of a divided bi-spaced aberrant SMC of Zea mays formed after caffeine treatment. Although the daughter wall exhibits very irregular profiles in different depths, in some planes it enters the SC space and forms a SC-like wall region (arrows in e and f). Note also the preferential fusion of the daughter wall with the margins of the incomplete SC wall of the previous division (arrows in c and d). N, nucleus (from Apostolakos & Galatis, 1987). found that the nucleus moves towards the site of pressure application (Fig. 5). Regardless of the nature of the inductive stimulus, between GMC and the SMCs, between the former and the SCs as well as between the young GCs and SCs, information is probably exchanged to ensure their coordinated function and differentiation. AFs might be implicated, not only in the transduction of the inducing stimulus polarizing the SMCs (see section II.3), but also in cell-to-cell communication through the cell wall and then across plasmalemma or through plasmodesmata. The lateral walls of Zea mays GMCs display numerous plasmodesmata arranged in primary pit fields (Galatis, 1982). The actomyosin system is probably involved in the function of plasmodesmata. Actin, myosins and other actin-related proteins have been localized in the plasmodesmata of several plants. Moreover, reliable experimental data suggest that the actomyosin system regulates the communication between neighbouring cells through plasmodesmata (Crawford & Zambryski, 1999; Heinlein, 2002; Volkmann et al., 2003). 5. Cytoskeleton and final cell plate alignment The alignment of the cell division plane in stomatal cells is a strictly controlled process in which the PPB region plays a crucial role. At this region a mechanism functions that attracts or guides the expanding cell plate edges to fuse with the underlying wall (Gunning, 1982; Wick, 1991a,b; Mineyuki, 1999; Pickett-Heaps et al., 1999; Cleary, 2000; Smith, 2001). Findings showing that the PPB region controls the final cell plate arrangement are: first the curved growth of the cell plate in asymmetrically dividing stomatal cells in order to meet the parent cell wall at the PPB region (among others see Pickett-Heaps & Northcote, 1966; Galatis & Mitrakos, 1979; Busby & Gunning, 1980); second the reorientation of the phragmoplast-cell plate system in telophase/cytokinetic GMCs of monocotyledons (Palevitz & Hepler, 1974a,b; Palevitz, 1986; Mineyuki et al., 1988; Cleary & Hardham, 1989) and in some SMCs of Tradescantia virginiana (Pickett-Heaps et al., 1999) to find the PPB region; and third the involvement of two PPB regions in the control of the final cell plate alignment in the double polarized SMCs of Triticum spp. (Galatis et al., 1983a; Fig. 3). Moreover, the plane of divisions in the stomatal pathway is sometimes controlled by astonishing accuracy. This is clearly seen in GMCs of Leguminosae spp. (Galatis & Mitrakos, 1980; Galatis et al., 1982) and Arabidopsis thaliana (Zhao & Sack, 1999), where the cell plate actually bisects the thickenings deposited on the wall lining the PPB region (see section III.1.2). In cytokinetic cells of higher plants, the edges of the advanced cell plate are connected to the PPB region by an AF system. These AFs seem to guide the cell plate to the PPB region interacting with some factor(s) residing in it by a mechanism in which myosins are involved (Verma, 2001; New Phytologist (2004) 161:

7 Review 619 Fig. 7 Diagrams to illustrate the cortical MT organization below the periclinal walls during morphogenesis of the stomatal complexes in Lolium (a o) and in a mature stoma of Avena (p). The lines denote MTs. (a d) Interphase MT band (MT-IB) in a GMC and preprophase MT band (MT-PPB) in SMCs. (e) Radial MT array in an advanced interphase GMC. (f h) MT-PPBs at different stages of organization in GMC and radial MT arrays in SCs. (i l) MT organization under the external periclinal wall (E) and the internal periclinal wall (I) in GCs and SCs of young stomata complexes until the stage of the kidneylike GCs (l). (m, n) MT arrangement during elongation of the stomatal complex. (o, p) MT organization in mature stomatal complexes of Lolium (o) and Avena (p) (modified from Cleary & Hardham, 1989). Hepler et al., 2002; Molchan et al., 2002). The disturbance of cell plate aligment in stomatal cells by cytochalasin B and D treatment (Palevitz & Hepler, 1974b; Palevitz, 1980; Cho & Wick, 1990, 1991; Gallagher & Smith, 1999; Pickett- Heaps et al., 1999) strengthens this view (Wick, 1991a,b; Pickett-Heaps et al., 1999; Cleary, 2000; Smith, 2001). Study of dividing SMCs of grasses showed that the mechanism functioning in the PPB region guides or attracts the cell plate only when its edges reach a certain distance from it (Galatis et al., 1984a,b). This is possible when the spatial organization of the cytokinetic protoplast is proper, that is the PPB region is not masked by large organelle(s) and the cell plate grows on a plane almost parallel to the PPB region. Otherwise, a portion of the cell plate may diverge and fuse with the parent walls far from the PPB region, forming triangular SCs or SCs with more complicated shapes (Galatis et al., 1984a,b). These conditions are established during SMC polarization by: first the placement of the nucleus close to the inducing GMC; second the proper organelle disposition; third the stabilization of the one mitotic spindle pole close to the GMC; and fourth the alignment of the mitotic spindle axis more or less transversely to the PPB plane (Galatis et al., 1983a, 1984a,b). In vivo study of Tradescantia virginiana SMCs revealed that the anchored spindle pole acts as a pivot point for the spindle. The other spindle pole swings around during cell division placing the one edge of the cell plate in close proximity to the PPB region. In these SMCs, realignment mechanism(s) function to correct cell plate arrangement, when one edge of the cell plate is in close proximity to the PPB region (Cleary, 2000; Pickett-Heaps et al., 1999). Factors affecting the plane of SMC division in normal Triticum spp. protoderm are the size of the SMC and the arrangement of its transverse walls in relation to the GMC, and mainly the function of two or more polarizing stimuli from different directions (Galatis et al., 1983a, 1984b). It is interesting that the shape of the atypical SCs in Triticum spp. often changes in space. In median paradermal planes, where the SC nucleus masked the PPB region, the SCs display a triangular form, while in external planes where the PPB region was approachable they exhibit the typical lens-like shape (Galatis et al., 1984b). Moreover, in caffeine-produced bi-spaced SMCs of Zea mays, atypical SCs are formed because wall strips prevent the cell plate from meeting the PPB region (Apostolakos & Galatis, 1987, Fig. 6). When in these cells the cell plate edge approaches the PPB region, it is guided to form a SC wall portion inside the wall strips of the aborted cell (Fig. 6e,f). According to Cleary (1995, 2000; see also Pickett-Heaps, 1969a) the curved growth of the cell plate in SMCs is controlled by interactions between the phragmoplast MTs and AFs with the SC nucleus. As a result, the cell plate is pulled around the nucleus. This hypothesis does not explain all cases. Often, the nascent cell plate grows on one plane and curves after its emergence from the interzonal region, when it faces the PPB This is very clear in the atypical SCs formed in the double polarized SMCs (Fig. 3c,d, see also Galatis et al., 1983a) and in SMCs divided during centrifugation (Galatis et al., 1984a). In some asymmetrically dividing stomatal cells, apart from the PPB region, there are other cortical sites, affecting the New Phytologist (2004) 161:

8 620 Review Fig. 8 (a, b) Light micrographs of paradermal sections of Zea mays leaves treated with colchicine (a) and caffeine (b). The arrows in (a) point to aberrant GMCs (compare with Figure 2), while the arrow in (b) marks an aberrant dumbbell-like stoma, which lacks a VW but displays a canal-like region. Bar, 10 µm. (c) TEM micrograph showing a median transverse section of an aberrant dumbbell-like stoma of Zea mays produced by caffeine treament. The absence of a VW does not prevent the canal formation and the deposition of the thickenings on the periclinal walls (arrows). Bar, 2 µm (from Galatis & Apostolakos, 1991). cell division plane. In three Anemia species that form floating stomata, the cell wall separating the GMC has a funnel-like shape and fuses with the periclinal walls of the mother cell only. The GMC progenitors are double polarized. A circular MT-PPB abutted on the external periclinal wall predicts the sites of fusion of the cell plate with the underlying wall regions. At the same time, a limited cortical region abutted on the internal periclinal wall outlining a minute intercellular space, defines the site of the cell plate fusion with the internal periclinal wall (Galatis et al., 1986). Cortical as well as endoplasmic MTs converge on this cortical region at preprophase. Moreover, cortical regions far from the PPB region, which display MTs during preprophase, locally affect the plane of cell division in protodermal cells of the liverwort Marchantia paleacea involved in air pore and air chamber development (Apostolakos & Galatis, 1985b,c) and in caffeine-produced bi-spaced SMCs of Zea mays (Apostolakos & Galatis, 1987). These sites guide or attract the cell plate to fuse with the underlying wall areas. Therefore, in double - and multipolarized cells, the cell plate does not fuse with the parent wall regions predicted Fig. 9 (a) TEM micrograph of a transversely sectioned GMC of Asplenium nidus being at an advanced interphase stage. The arrow indicates the constriction at the middle of the internal periclinal wall and the arrowhead the thinned median region of the external periclinal wall. Bar, 2 µm. (b, c) Optical sections through the cortical cytoplasm adjacent to the external periclinal wall of GMCs of Asplenium nidus after tubulin immunolabeling (b) and AF staining with rhodamine-phalloidin (c). Bar, 10 µm (a and c from Apostolakos et al., 1997). by the PPB only, but, following complicated but predictable paths, separates daughter cells of aberrant shape. III. Cytoskeleton and stomatal cell shaping 1. GMC shaping GMCs of the dumbbell-shaped stomata The newly formed GMCs of the dumbbell-shaped stomata are hexahedra with narrow lateral walls, which before symmetrical division expand appreciably (Fig. 7a h). The advanced GMCs are usually constricted at their mid-transverse plane, while their polar ends are swollen (Figs 2 and 7). In paradermal sections, the lateral walls appear curved inwards, while the periclinal ones exhibit a slight median transverse groove. The GMCs of grasses bear a well-organized interphase MT band lining the mid-region of the lateral and periclinal New Phytologist (2004) 161:

9 Review 621 Fig. 10 MT-immunolocalization (a, b) and AF staining with rhodamine-phalloidin (c, d) in Asplenium nidus stomata at an early stage of their morphogenesis. (a, c) Optical sections through the cortical cytoplasm below the external periclinal wall (b, d) optical sections through a median plane. The arrowheads in (b) and (d) mark the site of the internal stomatal pore. Bar, 10 µm. (a c) (from Apostolakos & Galatis, 1999); (d) (from Apostolakos & Galatis, 1998). walls (Galatis, 1974, 1982; Busby & Gunning, 1980; Cho & Wick, 1989; Cleary & Hardham, 1989; Mullinax & Palevitz, 1989, Fig. 7a d). This is established at an early interphase stage transversely to the axis of the stomatal row and exists until preprophase, when it is replaced by the MT-PPB (Fig. 7a h). A few MTs line the transverse walls anticlinally. By contrast, the polar regions below the periclinal walls are traversed by a relatively significant number of MTs. Initially, they are randomly oriented but later are seen to converge on the interphase MT band region (Galatis, 1982; Cleary & Hardham, 1989, Fig. 7a d). Externally to the interphase MT band, an identical CM band is deposited (Galatis, 1982). This allows the elongation of the GMCs parallel to the axis of the stomatal row but also results in the appearance of a median GMC constriction preventing the cell bulging locally. The advanced interphase GMCs of Zea mays display numerous endoplasmic MTs, which diverge from the mid-region of the interphase MT band below the periclinal walls and enter the endoplasm. These MT arrays persist in the preprophase GMCs and are related to the spatial arrangement of plastids, endoplasmic reticulum (ER) and the nucleus that becomes ellipsoidal (Galatis, 1982). The above data and the fact that colchicine affects GMC shaping (Fig. 8a) provide evidence that this process is MT-controlled. In early preprophase GMCs, the interphase MT band disintegrates, while the MTs lining the periclinal walls assume temporarily a radial arrangement converging on their midregion (Cho & Wick, 1989; Cleary & Hardham, 1989; Mullinax & Palevitz, 1989, Fig. 7e). At preprophase, a wide MT-PPB forms, transversely to the preceding interphase MT band (Pickett-Heaps & Northcote, 1966; Galatis, 1974, 1982; Busby & Gunning, 1980; Cho & Wick, 1989; Cleary & Hardham, 1989; Mullinax & Palevitz, 1989, Fig. 7f h). The above observations show that in interphase/preprophase transition the cytoplasm adjacent to the mid-region of the periclinal walls of the grass GMCs is the site of convergence of cortical and endoplasmic MTs. The median constriction of the GMCs of grasses establishes conditions favouring the formation of the GC canal (Fig. 1c). Some GMCs of Zea mays affected by colchicine at an advanced interphase stage differentiate into persistent GMCs exhibiting a bone-like shape. Moreover, in the same plant the complete inhibition of VW formation by caffeine treatment does not prevent the formation of a narrow, canal-like region in the forming aberrant stomata (Galatis & Apostolakos, 1991, Fig. 8b,c). The above suggests that the grass GC morphogenesis starts at the GMC stage and that the presence of a VW is not a prerequisite for it (Galatis, 1982; Galatis & Apostolakos, 1991). In interphase and preprophase GMCs of Secale cereale (Cho & Wick, 1990, 1991) AF bundles oriented transversely to the stomatal row axis run through the cortical cytoplasm. AF arrays resembling an interphase MT band and an AF-PPB have not been found. AF patches line the whole surface of the lateral walls in advanced interphase, mitotic and cytokinetic GMCs of the same plant. These are probably involved in stabilization of the mitotic spindle poles (Cho & Wick, 1990, 1991) or in the correct cell plate realignment during cytokinesis (Cleary, 2000). According to the current knowledge the cortical AFs are not, at least directly, involved in GMC shaping. GMCs of the elliptical stomata Detailed information on GMC shaping of the elliptical stomata is available only for the fern Asplenium nidus (Apostolakos et al., 1997). The GMCs of this plant undergo characteristic, MT-patterned, morphogenetic changes. The advanced interphase GMCs display a round form in surface view, which in median transverse planes becomes semilobed (Fig. 9a) by a constriction at the midregion of the internal periclinal wall and the two lateral anticlinal ones. The external periclinal wall is lined by a radial MT array (Fig. 9b), which converges on its thin mid-region (Fig. 9a), followed by deposition of similarly oriented CM arrays. The latter dictates the tangential expansion of the external periclinal wall, promoting GMC rounding. Moreover, a U-like MT bundle lines the site of future cell isthmus. Externally of this bundle a U-like CM bundle is deposited. The latter, preventing the increase of cell diameter on its plane, results in the assumption of the semilobed shape by the GMCs (see also Panteris et al., 1993b). Although in Asplenium nidus GMCs the cortical AFs form arrays similar to those of New Phytologist (2004) 161:

10 622 Review MTs (Fig. 9c; cf. Fig. 9b), they are not involved, at least not directly, in GMC morphogenesis (Apostolakos et al., 1997). In this plant, basic structural features of GCs are established at the GMC stage (Apostolakos et al., 1997; Apostolakos & Galatis, 1999). Thus, in elliptical stomata GC morphogenesis seems also to commence at the GMC stage (Galatis & Mitrakos, 1979; Galatis et al., 1982; Zhao & Sack, 1999). The assembly of radial MT and AF systems below the periclinal walls in the advanced interphase GMCs of the elliptical stomata is a rather general phenomenon. Radial MT arrays form in Selaginella spp. (Cleary et al., 1992) and Allium cepa (Mineyuki et al., 1989) GMCs, while radial AFs line the periclinal walls of Tradescantia virginiana GMCs (Cleary & Mathesius, 1996, Fig. 4b). MT-PPBs form in GMCs of the elliptical stomata of monocotyledons (Palevitz & Hepler, 1974a; Mineyuki et al., 1989), dicotyledons (Galatis & Mitrakos, 1979; Galatis et al., 1982; Kazama & Mineyuki, 1997; Zhao & Sack, 1999), the lower tracheophyte Selaginella spp. (Cleary et al., 1992) and in the ferns Azolla spp. and Asplenium nidus (Busby & Gunning, 1984; Apostolakos et al., 1997). The misorientation of the VW induced by red light in Cucumis sativus GMCs is predicted by a proper MT-PPB alignment (Kazama & Mineyuki, 1997). An exception to the above is the case of GMCs of the moss Funaria hygrometrica, which lack a MT-PPB and form an incomplete VW (Sack & Paolillo, 1985). Since some bryophyte cell types form incomplete MT-PPBs, that is incomplete MT rings (Apostolakos & Galatis, 1992 and literature therein), it should be re-examined whether GMCs of Funaria hygrometrica really lack a MT-PPB or form an incomplete one. AF-PPBs are assembled in the GMCs of Selaginella spp. (Cleary et al., 1992) and Tradescantia virginiana (Cleary, 1995; Cleary & Mathesius, 1996; Cleary, 2000). In the latter plant, the AF-PPB is disintegrated at late prophase leaving an AF-depleted zone persisting in mitosis. In 21 Leguminosae species (Galatis & Mitrakos, 1979; Galatis et al., 1982) and in the Cruciferae species Arabidopsis thaliana (Zhao & Sack, 1999) the anticlinal GMC wall regions adjacent to the MT-PPB become locally thickened. These thickenings are distinct in the young GCs and probably set up mechanical forces promoting stomatal pore formation (Galatis et al., 1982). In particular, the prethickened ends of the GC dorsal walls as well as the VW thickenings (see section III.2.1) may serve to make these anticlinal wall regions more resistant to expansion. As in stomatal movement they possibly result in the bending of the VW, thus facilitating the schizogenous opening of the stomatal pore (see section IV). The PPB region in GMCs of Leguminosae shows numerous coated regions on the plasmalemma and numerous coated vesicles, observations suggesting the function of a local endocytotic route at this site (see also Mineyuki et al., 2003). It should be noted here that local wall thickenings emerge at particular wall sites adjacent to the PPB region in colchicine affected SMCs of Zea mays (Galatis et al., 1984a). Besides, wall strips emerge in the PPB region of some caffeine-affected SMCs and GMCs of grasses (Pickett-Heaps, 1969b; Apostolakos & Galatis, 1987; Galatis & Apostolakos, 1991). They often grow inwards and rarely completely divide the affected cell (Apostolakos & Galatis, 1987). The above results show that the PPB region in stomatal cells has a complicated function, resulting from the establishment of a particular polarity in dividing cells (Fowler & Quatrano, 1997; Mineyuki, 1999; Pickett-Heaps et al., 1999; Cleary, 2000, 2001). Apart from its implication in the final cell plate alignment (see section II.5), under favourable conditions it promotes local wall deposition, and according to Mineyuki & Gunning (1990) the maturation of the daughter cell wall. 2. GC shaping Kidney-shaped GCs The differentiating as well as the mature kidney-shaped GCs are characterized by the presence of unequal wall thickenings and highly ordered CM arrays in their walls (Palevitz, 1981a, 1982; Sack, 1987). The periclinal walls contain radial CM arrays (Fig. 1b), converging on the rims of the stomatal pore (Ziegenspeck, 1938/39, 1944, Volz, 1952; Singh & Srivastava, 1973; Palevitz & Hepler, 1976; Galatis & Mitrakos, 1980; Galatis et al., 1983b; Sack & Paolillo, 1983b). The CMs in the ventral and dorsal GC walls are anticlinally oriented. The radial CMs are of fundamental importance, not only for GC shaping (Aylor et al., 1973) but also for stomatal movement (Meidner & Mansfield, 1968). The highly oriented deposition of the CM arrays in the GC walls occurs externally to identical cortical MT arrays. Each young GC displays: first two radial MT arrays underneath the periclinal walls focused on the cortical cytoplasm abutting on the junction of the mid-region of the VW with the periclinal walls (Fig. 10a); second an anticlinal MT bundle adjacent to the middle region of the VW, which pairs the focal regions of the radial MT arrays (Fig. 10b); and third anticlinal MTs more or less symmetrically distributed along the dorsal walls (Landré, 1969; Singh & Srivastava, 1973; Galatis, 1974; Palevitz & Hepler, 1976; Sanchez, 1977; Doohan & Palevitz, 1980; Galatis & Mitrakos, 1980; Galatis et al., 1983b; Sack & Paolillo, 1983a; Busby & Gunning, 1984; Davis & Gunning, 1992; Cleary et al., 1993; Apostolakos & Galatis, 1999). In GCs of Asplenium nidus, apart from the above MT arrays, periclinal MTs line the polar VW ends, where local wall thickenings displaying periclinal CMs emerge (Apostolakos & Galatis, 1999). Moreover, in advanced morphogenetic stages of Selaginella spp. GCs the MTs below the internal periclinal wall lose their radial organization and form concentric circles around the stomatal pore (Cleary et al., 1993). The asymmetrical organization of the MT arrays under the GC walls is the first structural event of GC differentiation (Galatis, 1974; Galatis & Mitrakos, 1980; Galatis et al., 1983b). New Phytologist (2004) 161:

11 Review 623 Local thickenings emerge along the whole length of the mid-region of the VW lined by the MT bundle as well as at the junction of the mid-region of the periclinal walls with the VW, before the opening of the stomatal pore (reviews by Palevitz, 1981a, 1982; Sack, 1987). Considering the consistency of coalignment between MT and CM arrays in stomatal cells, recent data confirming the MT involvement in CM orientation (Baskin, 2000, 2001; Burk & Ye 2002; Zhong et al., 2002) and the effects of MT depolymerization in GC shaping (Palevitz & Hepler, 1976), it is assumed that: first the cortical MT arrays function as a prepattern of the kidney-shaped GC morphogenesis, defining the CM orientation at every stage of the process. The kidney-like as well as the dumbell-like GC shape is the outcome of a continuous interplay between the growing protoplast and the properly reinforced anticlinal and periclinal walls, which accurately define the position and the direction of their expansion. Second the radial MT arrays below the periclinal walls are the key elements of the kidney-shaped GC morphogenesis. The radial CMs, which are deposited externally to them, allow a tangential expansion of the periclinal walls forcing the GC to assume a kidney-like shape. Third the paired radial MT arrays are implicated in the deposition of local wall thickenings. In differentiating kidney-shaped GCs, the cortical AFs form highly ordered arrays very similar to those of the cortical MTs (Cleary et al., 1993; Cleary & Mathesius, 1996; Apostolakos & Galatis, 1999). For example, the differentiating GCs of Asplenium nidus display radial AF arrays paired with an anticlinal AF bundle coexisting with those of MTs (Apostolakos & Galatis, 1999, Fig. 10c,d). Experimental work with cytochalasin B and C showed that the cortical AF arrays are not involved in the establishment of MT arrays or in GC morphogenesis (Palevitz & Hepler, 1976; Palevitz, 1980; Marc & Palevitz, 1990). Although their role in GC morphogenesis is ignored, similarly to other plant cell types (Hepler et al., 1990), they may control the cortical ER distribution. The ER in differentiating GCs is well developed and preferentially gathered near to the emerging wall thickenings (Galatis et al., 1983b; Sack & Paolillo, 1983a; Palevitz & Hodge, 1984). Considering the AF function in mature stomata (see section VII.1), it may be also suggested that they are involved in the massive entrance of ions in the young GCs, a phenomenon observed in Allium cepa (Palevitz & Hepler, 1976; Palevitz, 1981a, 1982). This activity contributes to the increase of protoplast volume, which is necessary for the assumption of the kidney-like shape by the GCs and the stomatal pore opening. Unlike in other plant cell types, the cortical MT and AF arrays persist in mature elliptical stomata, an observation implying their involvement in stomatal function (see section VII). Dumbbell-shaped GCs The mature dumbbell-shaped GCs have a narrow central canal region with intensely thickened periclinal walls and two thin-walled bulbous ends (Fig. 1c). In these cells, the CMs diverge from the rims of the canal towards the bulbous ends, while those of the periclinal walls of the canal tend to be longitudinal (Ziegenspeck, 1938/39; Galatis, 1980; Palevitz, 1981a; Fig. 1c). Caffeine-affected stomata of Zea mays, which lack a VW, tend to form a canal and to assume a dumbbell-like shape (Galatis & Apostolakos, 1991, Fig. 8b,c). Therefore, the VW is not necessary for the dumbbellshaped GC morphogenesis, which, as in the kidney-shaped GCs, is controlled by deposition of proper CM arrays in their walls (Galatis, 1974, 1980; Palevitz, 1981a, 1982; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989). Considering all the available information, it becomes clear that there are two distinct stages of dumbbell-shaped GC morphogenesis: first the transient assumption of a more or less kidney-like shape, which keeps pace with stomatal pore opening (Fig. 7b l) and second the integrated and highly controlled GC and SC elongation during which the stomatal pore elongates and the GC canal forms (Galatis, 1980; Palevitz, 1981a, 1982; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989, Fig. 7m p). At the first stage, the GCs display the MT (Fig. 7i l) and CM organization and the wall thickenings seen in the differentiating kidney-like GCs (Galatis, 1980; Palevitz, 1981a, 1982; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989). In this case, also radial MT systems determine the temporary kidney-like GC shape. The dumbbell-like GC shaping actually commences after completion of the stomatal pore opening (Kaufman et al., 1970; Srivastava & Singh, 1972; Galatis, 1980; Palevitz, 1981a, 1982; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989). The ventral and lateral walls of the GCs, as well as the anticlinal ones of the SCs are able to expand along their whole length, since they contain anticlinal CMs. By contrast, the periclinal walls of both cell types are able to elongate at their mid-region, where the CMs are nearly trasverse to the VW (Galatis, 1980). At the initiation of transition from the kidney-like to the dumbbell-like morphogenetic stage, the MTs adjacent to the internal periclinal GC wall lose their typical radial arrangement. New cortical MTs appear at the expanding mid-region of the GCs aligned transversely to their axis (Cleary & Hardham, 1989; Galatis & Apostolakos, 1991) as well as in the mid-region of the adjacent SCs (Cho & Wick, 1989; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989, Fig. 7k). The internal periclinal GC wall is first reinforced by transverse CMs. Similar changes in MT organization occur shortly after, at the mid-region of the external periclinal GC wall (Cleary & Hardham, 1989; Palevitz & Mullinax, 1989). All changes in cortical MT arrangement in the periclinal GC faces are followed by similar changes of the depositing CMs. In elongating GCs, the MTs fan out from the margins of the GC canal towards the bulbous ends of the GCs (Fig. 7m p). The cortical MTs underneath the expanding and intensely thickening periclinal regions of the canal, initially display a zig-zag arrangement (Fig. 7m,n) but finally become New Phytologist (2004) 161:

12 624 Review Fig. 11 (a) Light micrograph of a Zea mays stoma exposed to a prolonged colchicine treatment. Its morphogenesis has been blocked at the kidney-like GC stage. Bar, 10 µm. (b) Mature elliptical stoma of Zea mays taken from the very tip of a coleoptile as it appears with DIC optics. Bar, 10 µm. the two opposite radial MT arrays are paired with an anticlinal MT bundle (Palevitz & Mullinax, 1989). Therefore, all four cells of a young dumbbell-shaped stomatal complex display paired radial MT systems (Fig. 7i,j). If these MT systems are followed by deposition of similar radial CM arrays in the periclinal SC wall, the SC is prevented from elongating, but is forced to assume temporarily a lens-like shape in order to codifferentiate with the GCs. As it has been already noted, during GC elongation, the MTs under the mid-region of the periclinal SC walls become transverse to the stomatal axis. Such a CM orientation allows the coextension of the SC with the GCs. Finally, the cortical MTs in SCs become axially oriented as in Avena sativa (Palevitz & Mullinax, 1989, Fig. 7p) or remain transverse like in Lolium rigidum (Cleary & Hardham, 1989, Fig. 7o). The above show that the morphogenesis of the lens-shaped SCs in grasses is highly coordinated with that of the GCs. axial (Fig. 7o,p), always anticipating that of the depositing CMs (Kaufman et al., 1970; Galatis, 1980; Palevitz, 1981a, 1982; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989). Stomatal elongation is highly coordinated, both temporarily and positionally, between the GCs, between the GCs and the adjacent SCs and between the stomatal complex and the surrounding leaf cells. When the leaf elongation is inhibited or the MTs are disintegrated, the kidney-shaped grass GCs fail to assume a dumbbell-like shape (Fig. 11a). This is also the case for Zea mays stomata at the tip of mature coleoptiles, which remain elliptical, because in this region the elongation is diminished (Fig. 11b). Moreover, the cyperaceous stomata grown in the field are short and display radial CMs in the periclinal walls, while those developed in the glasshouse are more elongated, assume a rather typical dumbbell-like shape and show axial CMs in the canal region (Mishkind et al., 1981). Thus, the elongation is essential for the dumbbellshaped GC morphogenesis. In summary, it is reasonable to assume that the acquisition of a temporary kidney-like shape by GCs is a prerequisite for the dumbbell-like GC morphogenesis, that is the radial CM systems are of critical importance for the shift from the kidney-like to dumbbell-like pattern of GC morphogenesis. 3. Subsidiary cell shaping The available information on SC shaping is limited to the dumbbell-shaped stomata. Tubulin immunolabeling revealed definite shifts of MT organization in SCs of grasses at particular stages of stomatal development (Cho & Wick, 1989; Cleary & Hardham, 1989; Palevitz & Mullinax, 1989; Palevitz, 1991). The young SC displays transiently radial cortical MT systems below the periclinal walls, converging on the junction of their mid-region with the lateral wall of the GMC (Fig. 7g j). In each SC, the sites of convergence of IV. Stomatal pore formation One of the main tasks of GC morphogenesis is stomatal pore formation, a phenomenon concomitant to GC shaping that follows the deposition of local wall thickenings at the midregion of the VW and at its junctions with the periclinal walls. This starts from the external and/or the internal periclinal walls and proceeds inwards (Stevens & Martin, 1978; Galatis & Mitrakos, 1980; Galatis, 1980; Galatis et al., 1983b; Sack & Paolillo, 1983c; Sack, 1987). Stomatal pore formation involves two processes: the weakening of the middle lamella of the VW, and the application of mechanical forces to disrupt the periclinal walls and to separate the VW partners of the GC pair at the stomatal pore site. The middle lamella of the young VW of some angiosperms appears electron transparent in TEM micrographs (Singh & Srivastava, 1973; Galatis & Mitrakos, 1980; Galatis, 1980; Davis & Gunning, 1992; Zhao & Sack, 1999). It lacks pectic polysaccharidic materials, giving a negative reaction to Thiery s test that is specific for insoluble polysaccharides (Galatis, 1980; Galatis & Mitrakos, 1980). This electron transparency may also reflect callose deposition (Palevitz, 1981a; Davis & Gunning, 1992; Majewska-Sawka et al., 2002). The absence of pectic substances diminishes the adhesion between the VW partners, facilitating their separation. If callose is present, it can be rapidly degraded. However, there are stomata in which the middle lamella of the VW in TEM micrographs is typical in appearance. In these stomata, enzymatic hydrolysis of the middle lamella may be necessary, although no evidence of hydrolysis has been presented so far (for literature see Sack, 1987). The mechanical forces that locally separate the GCs are generated in the course of the assumption of the kidney shape by the GCs. They are applied to the thickened region of the VW and act as follows: the radial CM arrays allow the tangential expansion of the periclinal walls and force the dorsal walls to become curved. The local wall thickenings along the New Phytologist (2004) 161:

13 Review 625 stomatal pore region prevent the deformation of the VW at its mid-region and promote the local separation of the VW partners, after the disruption of the overlying periclinal wall regions. Afterwards, the separated VW partners move further apart from each other, to complete the stomatal pore (Galatis & Mitrakos, 1980). The same mechanism functions in the dumbbell-shaped stomata where the stomatal pore opening keeps pace with the temporal acquisition of a more or less kidney-like shape by the GCs (Galatis, 1980; Palevitz, 1981a, 1982; Palevitz & Mullinax, 1989). The forces creating the stomatal pore are generated by the increase of the protoplasm and/or of the turgor. K + and Cl ion levels increase substantially in Phleum pratense and Zea mays GCs before stomatal pore formation (Palevitz, 1981a, 1982). In the moss Funaria hygrometrica (Sack 1983c, 1987) and in the fern Azolla spp. (Busby & Gunning, 1984) cuticular precursors are deposited in the middle lamella at the region where the stomatal pore opens. This accumulation probably weakens the binding properties of the middle lamella, facilitating VW separation (Sack, 1987). In these cases, stomatal pore formation also follows the deposition of the radial CM arrays in the periclinal walls, keeping pace with GC shaping. A different mode of stomatal pore formation functions in the ferns Adiantum capillus-veneris (Galatis et al., 1983b), Anemia mandioccana (Zachariadis et al., 1997), Asplenium nidus (Apostolakos & Galatis, 1998, 1999) and probably in Polypodium vulgare (Stevens & Martin, 1978). In these ferns the phenomenon commences in very young GCs at the middepth of the VW by the local movement of the adjacent plasmalemmata, apart from each other, while the periclinal walls remain intact. The VW at this stage is a thin membranous diaphragm, which does not display detectable middle lamella and wall materials and contains callose. Thus, a rudimentary internal stomatal pore is formed, which broadens towards the periclinal walls (Fig. 12a). The final stomatal pore opening is achieved by disruption of the periclinal wall portions covering the internal stomatal pore (Zachariadis et al., 1997; Apostolakos & Galatis, 1998, 1999). The initiation of the internal stomatal pore coincides with the organization of the anticlinal MT bundles along the middle of the VW and the colocalization of AFs in the same sites (Zachariadis et al., 1997; Apostolakos & Galatis, 1998, 1999, Fig. 10b,d). Treatment of Anemia mandioccana and Asplenium nidus stomata with anti-mt drugs (Zachariadis et al., 1997; Apostolakos & Galatis, 1998) and taxol (our unpublished data) inhibits stomatal pore opening. Therefore, the MTs in the above ferns seem to be directly involved, by an uknown mechanism, in the separation of the plasmalemmata during the internal opening of the stomatal pore. This mechanism, at least in Asplenium nidus, is Ca 2+ -dependent. The mycotoxin cyclopiazonic acid, which interferes with the establishment of the ER-dependent Ca 2+ gradients, inhibits the internal stomatal pore formation in Asplenium nidus (our unpublished data; Fig. 12b). Afterwards, forces generated during GC shaping disrupt the periclinal wall remnants covering the internal stomatal pore. Therefore, the cortical MTs are directly involved in stomatal pore formation in some ferns and indirectly in the majority of the plants via MT implication in GC morphogenesis. The AFs do not seem to play a particular role in this process in angiosperms, since cytochalasin B does not inhibit stomatal pore formation (Palevitz, 1980). The stomata form a pore even when a typical VW is absent. In the caffeine-formed aberrant stomata of Zea mays (Galatis & Apostolakos, 1991) and in the cyd1 mutants of Arabidopsis thaliana (Yang et al., 1999) wall thickenings are deposited at the free end of one of the VW strips. In these aberrant stomata a modified mechanism of stomatal pore formation functions. The wall thickenings display electron dense material, which is degraded to form a rudimentary nonfunctional stomatal pore (Galatis & Apostolakos, 1991). The tendency to form a pore is astonishing in caffeine-formed aberrant stomata, which completely lack a VW. In these stomata, wall thickenings are deposited on the mid-region of the periclinal walls, which often grow inwards to form a continuous wall column surrounded by MTs (Galatis & Apostolakos, 1991, Fig. 13a,b). Electron dense materials are deposited in the wall column (Fig. 13a,b), the dissolution of which results in the formation of a rudimentary pore (Fig. 13c). These findings support a function of VW formation in bringing into communication the two periclinal walls to form the stomatal pore. V. Substomatal cavity formation Substomatal cavity initiation precedes stomatal pore formation. These processes are substages of a major process, stomatal morphogenesis, and keep pace with the intercellular space formation in the subepidermal tissues. Although it has been reported that the substomatal cavity formation may result from the lysis of some cells below the stomata (DeChalain & Berjak, 1979), there is conclusive evidence from the study of various plants that it is the result of a gradual schizogenous process (Laroche et al., 1976; Steven & Martin, 1978; Galatis, 1980, 1982; Sack & Paolillo, 1983a,b,c; Galatis et al., 1986; Apostolakos et al., 1997). Mechanical forces generated from the differential expansion between the GMC and the underlying cells, seem to play the major role in the substomatal cavity formation, although some local lytic activity on the middle lamella of the separating walls, at least at their junctions, cannot be excluded (Sack, 1987). Substomatal cavity initiation in three species of Anemia (Galatis et al., 1986) and in Asplenium nidus (Apostolakos et al., 1997), keeps pace with the morphogenesis of the GMCs or their progenitor cells. In Anemia spp., the interphase GMC progenitors undergo a peculiar polarization expressed on its shape. The internal periclinal wall at the polar end of the cell is locally detached from the walls of the adjacent cells, while the cell bulges slightly outwards. Thus, a minute intercellular New Phytologist (2004) 161:

14 626 Review space is initiated at the polar end of the cell, which gradually develops into a substomatal cavity. The initial detachment of the walls is the consequence of the shaping of the GMC progenitor only. Cortical MTs as well as endoplasmic MTs converge on the internal periclinal wall region bordering the minute intercellular space (Galatis et al., 1986). Similarly to Anemia spp., in Asplenium nidus minute intercellular spaces open between protodermal and subprotodermal cell layers below the proximal polarized end of the GMC progenitors, which gradually develop into substomatal cavities (Apostolakos et al., 1997). The formation of the median constriction in Asplenium nidus GMCs (see section III.1.2) promotes the further wall detachment between the GMC and the subepidermal cells, contributing to the broadening of the substomatal cavity. Inhibition of GMC morphogenesis with anti-mt drugs blocks the initiation and development of the substomatal cavities (Apostolakos et al., 1997). The assumption of the lobed form by the subepidermal cells also contributes to the development of the substomatal cavities. The morphogenesis of the lobed mesophyll cells in grasses (Jung & Wernicke, 1990; Apostolakos et al., 1991; Wernicke & Jung, 1992) and ferns (Panteris et al., 1993c) is accurately controlled by a system of highly organized MT bundles. They control the deposition of identical CM systems externally to them, which in turn define the positions of the cell contrictions. The lobed cell morphogenesis is always coupled with intercellular space formation. In Asplenium nidus the broadening of the substomatal cavity is the result of the temporal and positional coordination of the GMC morphogenesis with that of the underlying cells. Frequently, the U-like MT bundle defining the position of the GMC constriction (see section III.1.2) is opposite to a MT bundle defining the position of a cellular constriction in the subepidermal cell (Apostolakos et al., 1997). This synchronous and opposite formation of constrictions between the GMC and the subprotodermal cells contributes to the substomatal cavity development. The position of the U-like MT bundles, in the differentiating semilobed protodermal cells of Adiantum capillus-veneris, seems to be determined by the subprotodermal cells (Panteris et al., 1993b, 1994). Therefore, the coordination of morphogenesis between protodermal and subprotodermal cell layer resulting in substomatal cavity formation should be a more general phenomenon among plants. Fig. 12 (a) TEM micrograph showing a median transverse profile of a young stoma of Asplenium nidus. The arrow shows the internal stomatal pore and the arrowheads the thinned region of the external periclinal wall. Bar, 1 µm (from Apostolakos & Galatis, 1998). (b) TEM micrograph of a young stoma of Asplenium nidus treated with cyclopiazonic acid in a median transverse section. The formation of the internal stomatal pore has been inhibited (compare with (a)). The arrowheads point to the thinned region of the external periclinal wall of the GC. Bar, 2 µm. VI. Stomatal complex morphogenesis in mutants The morphogenesis of stomatal complexes in mutants has been studied in Zea mays and Arabidopsis thaliana. In the first plant the mutations dcd1, dcd2, pan1 and brk1 disturb cell plate arrangement during asymmetrical divisions but not of the symmetrical ones. In particular, the dcd1 and dcd2 mutations affect the divisions in the GMC progenitors and in the SMCs, producing GMCs and SCs that are atypical in New Phytologist (2004) 161:

15 Review 627 size and form, while pan1 and brk1 affect the divisions in SMCs forming atypical SCs (Gallagher & Smith, 1999, 2000; Smith, 2001). In the asymmetrically dividing GMC progenitors and SMCs of dcd1 and dcd2 mutants the one anticlinal edge of the cell plate fuses with the parent wall at the PPB site, while the other diverges and meets the parent walls at unpredicted sites or remains free in the cytoplasm. In this way, triangular or more abnormal daughter cells are formed or the cytokinesis is not completed (Gallagher & Smith, 1999). In the doublemutants dcd1/dcd2, apart from the above-described cell plate disarrangements, the cell plate in SMCs may fuse with parent wall regions far from the PPB. Thus, lens-shaped cells not adjacent to GMCs or circular cells, appearing free inside SMCs in a paradermal view, are produced (Gallagher & Smith, 2000). The structural polarization of the GMC progenitors and SMCs in dcd1, dcd2 and dcd1/dcd2 mutants is not affected. In particular: first the nucleus is placed at the polar end of the cell, which is marked by a cortical AF patch; second the MT- PPB and the AF-PPB appear in the expected position; third the one pole of the spindle is linked with AFs with the cortical AF patch; and fourth the phragmoplast-cell plate system is connected with the PPB region and the cortical AF patch by AFs (Gallagher & Smith, 1999, 2000; Smith, 2001). Therefore, in the mutants as in the wild type, all the cellular conditions are fulfilled for the cell plate to meet the parent walls at the predetermined position. According to the above authors, the discordia mutations affect the AF-based mechanism, which guides the margins of the expanding cell plate to fuse with the parent walls at the PPB region. This conclusion is based on: first the similarity of the effects of mutations on cell divisions to those produced by a cytochalasin D treatment in the wild type; and second the finding that cytochalasin D treatment in dcd1 and dcd2 mutants increases the number of the atypically dividing SMCs (Gallagher & Smith, 1999). The cell plate misalignments in SMCs of the discordia mutants resemble those in some SMCs of wild type Triticum spp. seedlings (Galatis et al., 1984b). In the latter, the PPB region is unable to guide the expanding cell plate edges completely, since it is locally covered by larger organelles or the diverging cell plate edge grows far from it (see section II.5). Therefore, in these cells the AF systems involved in the final cell plate orientation seem to be functional because in planes Fig. 13 TEM micrographs of aberrant stomata of Zea mays formed after caffeine teatment. (a) Median transverse section of an aberrant stoma showing a wall column (arrow) which grew inwards from the middle of the external periclinal wall. This displays an electron dense material. Bar, 1 µm. (b) Median paradermal section of an aberrant stoma possessing a wall column (arrow). Bar, 1 µm (from Galatis & Apostolakos, 1991). (c) Median paradermal section of an aberrant stoma. A rudimentary stomatal pore has been formed at the middle of the wall column (arrow). Bar, 2 µm. New Phytologist (2004) 161:

16 628 Review where the developing cell plate approaches the PPB region it grows towards it (Galatis et al., 1984a,b). As in Triticum SMCs, the disturbance of the SMC division plane in discordia mutants may also be due to an inappropriate spatial organization of the dividing protoplast, as the result of an improper cell polarization. In pan1 and brk1 mutants, SMC polarization is completely inhibited or significantly disturbed (Gallagher & Smith, 2000; Smith, 2001; Frank & Smith, 2002). This view relies on the following observations: first that many SMCs of the brk1 mutants lack AF patches; second that the nuclear migration towards the inducing GMC is inhibited in many SMCs of both the above mutations; and third that about 25% of SMCs of the pan1 mutants do not form a PPB at the expected position laterally to the inducing GMC, but obliquely or transversely to the long SMC axis. In these cells, the phragmoplast-cell plate system traverses the cell diagonally or transversely, forming equal or unequal daughter cells, which, according to the criteria used by the authors, do not display features of SCs (Gallagher & Smith, 2000; Frank & Smith, 2002). Frank & Smith (2002) found that the BRK1 gene encodes a novel 8KD protein that may be involved in the AF-dependent cell polarization. However, in SMCs of the pan1 and brk1 mutants the nuclear behaviour, the PPB position, and the orientation of the mitotic spindle may predict a symmetrical and not an asymmetrical division. Although in grasses the cells of the rows, which function as SMCs, have completed their division cycle, they rarely undergo symmetrical divisions (Galatis et al., 1983a, 1984b). In the latter case, they are not competent to form a SC. Therefore it is possible that pan1 and brk1 mutations affect the time-course of the asymmetrical and symmetrical divisions in protodermal cell rows involved in stomatal development. Three mutations of Arabidopsis thaliana, the tmm, flp and sdd1, disorder the pattern of distribution of stomata inducing their grouping (Yang & Sack, 1995; Larkin et al., 1997; Berger & Altmann, 2000). They affect the position and the plane of asymmetrical divisions of the protodermal cells adjacent to preexistent stomata, yielding stomatal meristemoids in contact with them (Larkin et al., 1997; Geisler et al., 2000; Nadeau & Sack, 2003). The TMM gene encodes a leucinerich repeat-containing receptor-like protein that is cooperating with a subtilisin-like serine protease, which is encoded by the SDD1 gene (Berger & Altmann, 2000) and is involved in the control of the cell division plane (von Groll & Altmann, 2001; Serna & Fenoll, 2002; von Groll et al., 2002; Nadeau & Sack, 2003). The mutation of the TMM and SDD1 genes disturbs the pattern of stomatal distribution in epidermis (von Groll et al., 2002; Nadeau & Sack, 2002, 2003). TMM and SDD1 genes appear to function in a position-dependent signalling pathway that controls the plane of patterning divisions as well as the balance between stem cell renewal and differentiation in stomatal and epidermal development (Nadeau & Sack, 2002, 2003; von Groll et al., 2002). However, although the products of TMM gene seem to be related to the mechanism determining the plane of cell division in Arabidopsis thaliana protoderm, they do not control the nuclear position in the asymmetrically dividing cells (Geisler et al., 2003). It is not known whether the TMM or SDD1 gene affects PPB position or organization in these cells. In Arabidopsis thaliana the cytokinesis defective (cyd1) mutation affects the symmetrical division of the GMCs, disturbing cytokinesis. In cyd1 mutants aberrant stomata are formed, that in paradermal view display one or two incomplete VW strips opposite to one another or even they lack them completely (Yang et al., 1999). The free end of one of the VW strips becomes locally thickened and a stomatal pore is initiated. The effects of cyd1 mutation mimic those induced by caffeine treatment in Zea mays stomata (Galatis & Apostolakos, 1991). Like caffeine, cyd1 mutation may disturb cell plate stabilization, thus leading to partial or total inhibition of cytokinesis in GMCs. All the other phenomena of GC morphogenesis are carried out normally in the atypical stomata, even in the complete absence of a VW. VII. Cytoskeleton dynamics in functioning stomata 1. Actin filaments The mature kidney-shaped GCs are unique in that they are able to change the organization of their cortical cytoskeleton in response to external environmental factors, inducing stomatal movement. In stomata opened under white light, the cortical AFs below the periclinal walls are radially arranged around the stomatal pore (Fig. 14), an arrangement resembling that of MTs (Hwang et al., 2000). By contrast, in stomata closed in darkness or by other stimuli, the radial AFs are disintegrated (Kim et al., 1995; Eun & Lee, 1997, 2000; Hwang et al., 2000; Hwang & Lee, 2001). Long cortical and subcortical AF bundles displaying various orientations replace the radial AF arrays (Hwang & Lee, 2001; Fig. 14). These results have Fig. 14 Model of the paths of GC AF reorganization during stomatal closure activated by ABA treatment and the signal mediators involved therein (from Hwang & Lee, 2001). New Phytologist (2004) 161:

17 Review 629 been mainly derived from Commelina communis, using AF immunolocalization or AF staining by rhodamine-phalloidin in fixed cells or by microinjection of fluorescein isothyocyanatephalloidin into living GCs. The changes in cortical AF organization during stomatal movement have been also confirmed in vivo in Arabidopsis transgenic plants (WT/GFPmTn line) expressing a GFP fusion protein targeted to actin (Dong et al., 2001; Lemichez et al., 2001). Radial AF arrays have been also found in open stomata of Vicia faba and Nicotiana plumbaginifolia, but in these plants they are not so obvious compared to Commelina communis. Radial-more or less-af arrays have also been observed in GCs of Selaginella spp. (Cleary et al., 1993), Tradescantia virginiana (Cleary & Mathesius, 1996) and in Arabidopsis thaliana (wild plants, mutants and plants transformed with a GFP-mouse talin reporter; Kost et al., 1998; Eun et al., 2001). The diverse responses of the radial AF arrays to opening stimuli provide evidence that their organization in functioning GCs is not a result of stomatal movement. Thus: first circadian clock-induced opening of stomata is accompanied by the assembly of radial cortical AF arrays (Eun & Lee, 1997); second fusicoccin, a proton pump activator inducing excessive stomatal opening, inhibits the formation of radial AFs and disrupts the existing ones (Eun & Lee, 2000); and third hypotonic treatment of Vicia faba GCs, which promotes stomatal opening, induces AF disintegration (Liu & Luan, 1998). Besides, abscisic acid (ABA) treatment of Commelina communis and Arabidopsis thaliana, which induces stomatal closure, disrupts radial AF arrays rapidly (Eun & Lee, 1997; Hwang & Lee, 2001; Lemichez et al., 2001). Experimental work on Commelina communis, Vicia faba and Arabidopsis thaliana demonstrated that the cortical AFs operate as a signal mediator in moving GCs (Kim et al., 1995; Eun & Lee, 1997; Hwang et al., 1997; Liu & Luan, 1998; Dong et al., 2001; Lemichez et al., 2001; Hwang et al., 2000; Hetherington, 2001; Schroeder et al., 2001), a hypothesis also made in other plant systems (Staiger, 2000). In particular, the rapid disruption of the radial AF arrays in stomata after ABA treatment implies their early involvement in the signal transduction (Eun & Lee, 1997; Hwang & Lee, 2001; Lemichez et al., 2001). ABA is a potent stimulus of stomatal closing. It is significant that in the ABA-insensitive mutant abil 1 of Arabidopsis the cortical AF arrays are not disrupted after ABA treatment (Eun et al., 2001; Lemichez et al., 2001). The localization, of integrin-like proteins in the plasmalemma lining the dorsal walls of Vicia faba stomata probably explains the rapid response of the GC cytoskeletal elements to external stimuli (Zhang et al., 2001). Cytochalasin D treatment, which disrupts the radial AF arrays in GCs (Kim et al., 1995; Eun & Lee, 2000), promotes the stomatal pore opening, which is induced by white light and by circadian clock and enhances stomatal closure in the presence of ABA, under low external CO 2 and high K + concentration in the medium, conditions in which the stomatal closure does not easily occur. Besides, the AF-stabilizing agent phalloidin inhibits stomatal opening induced by white light and fusicoccin or circadian clock; it also inhibits the ABA induced stomatal closure (Hwang et al., 2000). These results further support the role of AFs as a signal mediator during stomatal movement and show that in the presence of cytochalasin D the GCs become more responsive to the stimuli, while phalloidin has the opposite effect. AFs are probably implicated in stomatal movements by regulation of activity and possibly the arrangement of ion channels in the GC plasmalemma (Kim et al., 1995; Hwang et al., 1997, 2000). Cytochalasin D, which promotes the lightinduced stomatal opening, potentiates the inward K + current Fig. 15 Model of the pathway by which AFs (red lines) modulate K + channel activity in GCs during stomatal movement. Purple squares: K + channels in a responsive state. Purple triangles: K + channels in an unresponsive state. Yellow flashes: hyperpolarizing membrane potential. Pink circles: actin binding proteins (from Plant Physiology, 115(2), 1997). New Phytologist (2004) 161: