SummaryThe morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed. New Phytologist (2005) 167 : 721-732© New Phytologist (2005)

Contents Summary000 Introduction000 Cytoskeleton and development of the stomatal complexes000 Cytoskeleton and stomatal cell shaping000 Stomatal pore formation000 Substomatal cavity formation000 Stomatal complex morphogenesis in mutants000 Cytoskeleton dynamics in functioning stomata000 Mechanisms of microtubule organization in stomatal cells000 Conclusions‐perspectives000 References000 Summary 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.

In this study, the effects of disturbance of the reactive oxygen species (ROS) homeostasis on the organization of tubulin cytoskeleton in interphase and mitotic root-tip cells of Triticum turgidum and Arabidopsis thaliana were investigated. Reduced ROS levels were obtained by treatment with diphenylene iodonium (DPI) and N-acetyl-cysteine, whereas menadione was applied to achieve ROS overproduction. Both increased and low ROS levels induced: (a) Macrotubule formation in cells with low ROS levels and tubulin paracrystals under oxidative stress. The protein MAP65-1 was detected in treated cells, exhibiting a conformation comparable to that of the atypical tubulin polymers. (b) Disappearance of microtubules (MTs). (c) Inhibition of preprophase band formation. (d) Delay of the nuclear envelope breakdown at prometaphase. (e) Prevention of perinuclear tubulin polymer assembly in prophase cells. (f) Loss of bipolarity of prophase, metaphase and anaphase spindles. Interestingly, examination of the A. thaliana rhd2/At respiratory burst oxidase homolog C (rbohc) NADPH oxidase mutant, lacking RHD2/AtRBOHC, gave comparable results. Similarly to DPI, the decreased ROS levels in rhd2 root-tip cells, interfered with MT organization and induced macrotubule assembly. These data indicate, for first time in plants, that ROS are definitely implicated in: (a) mechanisms controlling the assembly/disassembly of interphase, preprophase and mitotic MT systems and (b) mitotic spindle function. The probable mechanisms, by which ROS affect these processes, are discussed.

Treatment of root-tip cells of Triticum turgidum with 1 M mannitol solution for 30 min induces microtubule (Mt) disintegration in the plasmolyzed protoplasts. Interphase plasmolyzed cells possess many cortical, perinuclear and endoplasmic macrotubules, 35 nm in mean diameter, forming prominent arrays. In dividing cells macrotubules assemble into aberrant mitotic and cytokinetic apparatuses resulting in the disturbance of cell division. Putative tubulin paracrystals were occasionally observed in plasmolyzed cells. The quantity of polymeric tubulin in plasmolyzed cells exceeds that in control cells. Root-tip cells exposed for 2-8 h to plasmolyticum recover partially, although the volume of the plasmolyzed protoplast does not change detectably. Among other events, the macrotubules are replaced by Mts, chromatin assumes its typical appearance and the cells undergo typical cell divisions. Additionally, polysaccharidic material is found in the periplasmic space. Oryzalin and colchicine treatment induced macrotubule disintegration and a significant reduction of protoplast volume in every plasmolyzed cell type examined, whereas cytochalasin B had only minor effects restricted to differentiated cells. These results suggest that Mt destruction by hyperosmotic stress, and their replacement by tubulin macrotubules and putative tubulin paracrystals is a common feature among angiosperms and that macrotubules are involved in the mechanism of protoplast volume regulation.

Morphogenesis of sinuous epidertnal cells in leaves of the fern Aspleniiim nidus and the monocotyledonous Cyperus papyrus, petals of the dicotyledonous Begonia lucerna, and in-vitro-^nywn lea\'es of the fern Adiantum capillusveneris is controlled by the local differentiation of their wails. In all these cases wall pads, including radial cellulose microfibrils, are deposited at the junctions of the external periclinal wall with the anticlinal ones. Moreover, in Asplenium nidus, similar wall pads form at thejunctions of the internal periclinal wall with the anticlinal ones. The wall pads are connected to anticlinal cellulose mjcrofibril bundles running the whole depth of the anticlinal walls or part of it. This wail differentiaiion imposes a highly controlled cell wall expansion, a consequence of which is the waviness of the epidermal cell anticlinal walls. The pattern of wall reinforcement varies among different species, resulting in differences in the pattern of waviness. Cortical microtubule arrays mirror the orientated deposition of cellulose microtibrils in the epidermal cells.These tindings, derived from plants from diflerent major groups, show a common epidermal cell morphogenetic mechanism depending on radial cellulose microfibrils and cellulose micrnfibril bundles. The facts that {a) epidermal cell morphogenesis in Adiantum capiihis-reneris lea\es grown in Tiiro differs considerably from tbat of typical leaves and (h) petal epidermal cells in Begonia lucerna are sinuous, while Icat epidermal cells are not. suggest that this mechanism may be affected hy epigenetic factors.