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.

Plant cells divide their cytoplasmic content by forming a new membrane compartment, the cell plate, via a rerouting of the secretory pathway toward the division plane aided by a dynamic cytoskeletal apparatus known as the phragmoplast. The phragmoplast expands centrifugally and directs the cell plate to the preselected division site at the plasma membrane to fuse with the parental wall. The division site is transiently decorated by the cytoskeletal preprophase band in preprophase and prophase, whereas a number of proteins discovered over the last decade reside continuously at the division site and provide a lasting spatial reference for phragmoplast guidance. Recent studies of membrane fusion at the cell plate have revealed the contribution of functionally conserved eukaryotic proteins to distinct stages of cell plate biogenesis and emphasize the coupling of cell plate formation with phragmoplast expansion. Together with novel findings concerning preprophase band function and the setup of the division site, cytokinesis and its spatial control remain an open-ended field with outstanding and challenging questions to resolve.

Kinesins are versatile nano-machines that utilize variable non-motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin-12 orthologs, PHRAGMOPLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre-selected cell plate fusion site at the cell cortex. Here, we report on the spatio-temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine-tuned by its carboxy-terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule-associated protein MAP65-3/PLEIADE, a well-established microtubule cross-linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division.

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Aniline blue staining and callose immunolabeling revealed the deposition of significant callose quantities, in the form of fibrils, in the periclinal walls of guard cells (GCs) of stomata of the fern Asplenium nidus. The stomata that were at an early stage of differentiation displayed short callose fibrils at the junctions of the periclinal walls with the dorsal ones, which converged on the site of the future stomatal pore. In stomata being at an advanced stage of differentiation, callose fibrils were radially arranged around the stomatal pore, while in mature closed ones they were focused on the margins of the wall thickenings lining the stomatal pore. The pattern of the callose fibril organization resembled that of cellulose microfibrils in the same walls. Like the cellulose microfibrils, callose fibrils appeared coaligned with the underlying radial arrays of cortical microtubules (MTs). Moreover, the stomata treated with cellulose synthesis inhibitors (coumarin or dichlobenil) and those recovering from treatments with callose synthesis inhibitors (2-deoxy-D-glucose or tunicamycin) exhibited distinct radial callose fibril arrays. Cytochalasin B did not affect the organization of the radial callose fibril arrays. In contrast, oryzalin completely disturbed the pattern of callose deposition in the affected GCs. Therefore, the fibrillar callose orientation in the periclinal GC walls is probably controlled by MTs but not by actin filaments. The MTs seem to orient callose synthases in the plasmalemma, thus determining the fibrillar nature of callose deposits and their radial mode of arrangement. The cellulose microfibrils are not involved in the callose fibril alignment.

The data presented in this work revealed that in Zea mays the exogenously added auxins indole-3-acetic acid (IAA) and 1-napthaleneacetic acid (NAA), promoted the establishment of subsidiary cell mother cell (SMC) polarity and the subsequent subsidiary cell formation, while treatment with auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 1-napthoxyacetic acid (NOA) specifically blocked SMC polarization and asymmetrical division. Furthermore, in young guard cell mother cells (GMCs) the PIN1 auxin efflux carriers were mainly localized in the transverse GMC faces, while in the advanced GMCs they appeared both in the transverse and the lateral ones adjacent to SMCs. Considering that phosphatidyl-inositol-3-kinase (PI3K) is an active component of auxin signal transduction and that phospholipid signaling contributes in the establishment of polarity, treatments with the specific inhibitor of the PI3K LY294002 were carried out. The presence of LY294002 suppressed polarization of SMCs and prevented their asymmetrical division, whereas combined treatment with exogenously added NAA and LY294002 restricted the promotional auxin influence on subsidiary cell formation. These findings support the view that auxin is involved in Z. mays subsidiary cell formation, probably functioning as inducer of the asymmetrical SMC division. Collectively, the results obtained from treatments with auxin transport inhibitors and the appearance of PIN1 proteins in the lateral GMC faces indicate a local transfer of auxin from GMCs to SMCs. Moreover, auxin signal transduction seems to be mediated by the catalytic function of PI3K.

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