KINESIN-12E regulates metaphase spindle flux and helps control spindle size in Arabidopsis.

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.

Dual localized kinesin-12 POK2 plays multiple roles during cell division and interacts with MAP65-3.

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.

Plant Kinesin-12: Localization Heterogeneity and Functional Implications.

Kinesin-12 family members are characterized by an N-terminal motor domain and the extensive presence of coiled-coil domains. Animal orthologs display microtubule plus-end directed motility, bundling of parallel and antiparallel microtubules, plus-end stabilization, and they play a crucial role in spindle assembly. In plants, kinesin-12 members mediate a number of developmental processes including male gametophyte, embryo, seedling, and seed development. At the cellular level, they participate in critical events during cell division. Several kinesin-12 members localize to the phragmoplast midzone, interact with isoforms of the conserved microtubule cross-linker MICROTUBULE-ASSOCIATED PROTEIN 65 (MAP65) family, and are required for phragmoplast stability and expansion, as well as for proper cell plate development. Throughout cell division, a subset of kinesin-12 reside, in addition or exclusively, at the cortical division zone and mediate the accurate guidance of the phragmoplast. This review aims to summarize the current knowledge on kinesin-12 in plants and shed some light onto the heterogeneous localization and domain architecture, which potentially conceals functional diversification.

Phosphorylation of a p38-like MAPK is involved in sensing cellular redox state and drives atypical tubulin polymer assembly in angiosperms.

Reactive oxygen species (ROS) imbalance is a stressful condition for plant cells accompanied by dramatic changes in tubulin cytoskeleton. Here, evidence is provided that alterations in ROS levels directly interfere with the phosphorylation state of a p38-like MAPK in the angiosperms Triticum turgidum and Arabidopsis thaliana. Both oxidative stress generators and chemicals inducing ROS scavenging or decreasing ROS production resulted in the accumulation of a phospho-p46 protein similar to p38-MAPK. Importantly, the rhd2 A. thaliana mutants exhibited a remarkable increase in levels of phospho-p46. The presence of the p38-MAPK inhibitor SB203580 attenuated the response to ROS disturbance, prevented microtubule disappearance and resulted in a dramatic decrease in the number of atypical tubulin polymers. Moreover, in roots treated simultaneously with substances inducing ROS overproduction and others resulting in low ROS levels, phospho-p46 levels and the organization of tubulin cytoskeleton were similar to controls. Collectively, our experimental data suggest, for the first time in plants, that p46 functions as a putative sensor of redox state, the activation of which initiates downstream signalling events leading to microtubule disruption and subsequent assembly of atypical tubulin polymers. Thus, p46 seems to participate in perception of ROS homeostasis disturbance as well as in cellular responses to redox imbalance.

Arabidopsis pavement cell shape formation involves spatially confined ROPGAP regulators.

In many plant species, pavement cell development relies on the coordinated formation of interdigitating lobes and indentations. Polarity signaling via the activity of antagonistic Rho-related GTPases from plants (ROPs) was implicated in pavement cell development, but the spatiotemporal regulation remained unclear. Here, we report on the role of the PLECKSTRIN HOMOLOGY GTPase ACTIVATING PROTEINS (PHGAPS) during multipolar growth in pavement cell shape establishment. Loss of function in phgap1phgap2 double mutants severely affected the shape of Arabidopsis leaf epidermal pavement cells. Predominantly, PHGAPs interacted with ROP2 and displayed a distinct and microtubule-dependent enrichment along the anticlinal cell face and transfacial boundary of pavement cell indentation regions. This localization was established upon undulation initiation and was maintained throughout the expansion of the cell. Our data suggest that PHGAP1/REN2 and PHGAP2/REN3 are key players in the establishment of ROP2 activity gradients and underscore the importance of locally controlled ROP activity for the orchestrated establishment of multipolarity in epidermal cells.

IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation.

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.

Microtubule involvement in the deposition of radial fibrillar callose arrays in stomata of the fern Asplenium nidus L.

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.

Division Plane Establishment and Cytokinesis.

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.

Promoter analysis and functional implications of the selenium binding protein (SBP) gene family in Arabidopsis thaliana.

Selenium Βinding Protein (SBP, originally termed SBP56) was identified in mouse liver as a cytosolic protein that could bind radioactive selenium. SBPs are highly conserved proteins present in a wide array of species across all kingdoms and are likely to be involved in selenium metabolism. In Arabidopsis, the selenium binding protein (SBP) gene family comprises three genes (AtSBP1, AtSBP2 and AtSBP3). AtSBP1 and AtSBP2 are clustered in a head-to-tail arrangement on chromosome IV, while AtSBP3 is located on chromosome III. In this work, we studied the promoter activity of the Arabidopsis SBP genes, determined their tissue specificity and showed that they are differentially regulated by sodium selenite and sodium selenate. All three SBP genes are upregulated in response to externally applied selenium compounds and the antioxidant NAC selectively downregulates SBP2. Although the effect on SBP2 levels was the most prominent, in all cases, the concurrent exposure of plants to selenite and the antioxidant supressed the expression of the SBP genes. We provide evidence that (at least) SBP1 expression is tightly linked to detoxification processes related to oxidative stress, since it is downregulated in the presence of NAC in selenium-treated plants. Furthermore, our results suggest that SBP genes may participate in the mechanisms that sense redox imbalance.

Analysis of Phragmoplast Kinetics During Plant Cytokinesis.

In plants, the partitioning of daughter cells during cytokinesis is achieved via physical insertion of a membranous cell plate within the dividing parent cell. It is a cellular process of extensive protein secretion and membrane trafficking toward the plane of cell division and the cytoskeleton is an important facilitator of this process. A specialized cytoskeletal array termed phragmoplast expands centrifugally throughout cytokinesis and directs, mostly Golgi-derived vesicles that ultimately fuse to form the developing cell plate. The function of the phragmoplast in guiding cell plate synthesis has strongly motivated many scientists to monitor its dynamic behavior. In this chapter, we present an overview of basic principles and methods concerning the live imaging of cytokinetic plant cells using confocal laser scanning microscopy (CLSM) and the analysis of phragmoplast expansion.

ROS homeostasis as a prerequisite for the accomplishment of plant cytokinesis.

Reactive oxygen species (ROS) are emerging players in several biological processes. The present work investigates their potential involvement in plant cytokinesis by the application of reagents disturbing ROS homeostasis in root-tip cells of Triticum turgidum. In particular, the NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, and menadione that leads to ROS overproduction were used. The effects on cytokinetic cells were examined using light, fluorescence, and transmission electron microscopy. ROS imbalance had a great impact on the cytokinetic process including the following: (a) formation of atypical "phragmoplasts" incapable of guiding vesicles to the equatorial plane, (b) inhibition of the dictyosomal and/or endosomal vesicle production that provides the developing cell plates with membranous and matrix polysaccharidic material, (c) disturbance of the fusion processes between vesicles arriving on the cell plate plane, (d) disruption of endocytic vesicle production that mediates the removal of the excess membrane material from the developing cell plate, and (e) the persistence of large callose depositions in treated cell plates. Consequently, either elevated or low ROS levels in cytokinetic root-tip cells resulted in a total inhibition of cell plate assembly or the formation of aberrant cell plates, depending on the stage of the affected cytokinetic cells. The latter failed to expand towards cell cortex and hence to give rise to complete daughter cell wall. These data revealed for the first time the necessity of ROS homeostasis for accomplishment of plant cytokinesis, since it seems to be a prerequisite for almost every aspect of this process.

Deliberate ROS production and auxin synergistically trigger the asymmetrical division generating the subsidiary cells in Zea mays stomatal complexes.

Subsidiary cell generation in Poaceae is an outstanding example of local intercellular stimulation. An inductive stimulus emanates from the guard cell mother cells (GMCs) towards their laterally adjacent subsidiary cell mother cells (SMCs) and triggers the asymmetrical division of the latter. Indole-3-acetic acid (IAA) immunolocalization in Zea mays protoderm confirmed that the GMCs function as local sources of auxin and revealed that auxin is polarly accumulated between GMCs and SMCs in a timely-dependent manner. Besides, staining techniques showed that reactive oxygen species (ROS) exhibit a closely similar, also time-dependent, pattern of appearance suggesting ROS implication in subsidiary cell formation. This phenomenon was further investigated by using the specific NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, menadione which leads to ROS overproduction, and H2O2. Treatments with diphenylene iodonium, N-acetyl-cysteine, and menadione specifically blocked SMC polarization and asymmetrical division. In contrast, H2O2 promoted the establishment of SMC polarity and subsequently subsidiary cell formation in "younger" protodermal areas. Surprisingly, H2O2 favored the asymmetrical division of the intervening cells of the stomatal rows leading to the creation of extra apical subsidiary cells. Moreover, H2O2 altered IAA localization, whereas synthetic auxin analogue 1-napthaleneacetic acid enhanced ROS accumulation. Combined treatments with ROS modulators along with 1-napthaleneacetic acid or 2,3,5-triiodobenzoic acid, an auxin efflux inhibitor, confirmed the crosstalk between ROS and auxin functioning during subsidiary cell generation. Collectively, our results demonstrate that ROS are critical partners of auxin during development of Z. mays stomatal complexes. The interplay between auxin and ROS seems to be spatially and temporarily regulated.

RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility.

Ubiquitin mediated protein degradation constitutes one of the most complex post translational gene regulation mechanisms in eukaryotes. This fine-tuned proteolytic machinery is based on a vast number of E3 ubiquitin ligase complexes that mark target proteins with ubiquitin. The specificity is accomplished by a number of adaptor proteins that contain functional binding domains, including the WD40 repeat motif (WDRs). To date, only few of these proteins have been identified in plants. An RNAi mediated silencing approach was used here to functionally characterize the Arabidopsis thaliana ULCS1 gene, which encodes for a small molecular weight WDR protein. AtULCS1 interacts with the E3Cullin Ring Ligase subunit DDB1a, regulating most likely the degradation of specific proteins involved in the manifestation of diverse developmental events. Silencing of AtULCS1 results in sterile plants with pleiotropic phenotypes. Detailed analysis revealed that infertility is the outcome of anther indehiscence, which in turn is due to the impairment of the plants to accomplish secondary wall modifications. Furthermore, IREGULAR XYLEM gene expression and lignification is diminished in anther endothecium and the stem vascular tissue of the silenced plants. These data underline the importance of AtULCS1 in plant development and reproduction.

APRF1 promotes flowering under long days in Arabidopsis thaliana.

Arabidopsis thaliana flowering time mutants revealed the function of numerous genes that regulate the transition from vegetative to reproductive growth. Analyses of their loci have shown that many of them act as chromatin modifiers. In this study, a combination of molecular and genetic approaches have been implemented, to characterize the function of APRF1 (ANTHESIS POMOTING FACTOR 1) gene in A. thaliana and to investigate its role in plant development. APRF1 encodes for a low molecular weight nuclear WDR protein which displays functional homology to the Swd2 protein, an essential subunit of the yeast histone methylation COMPASS complex. Compared to WT plants, total loss-of-function aprf1 mutants exhibited shoot apical meristem (SAM) alterations and increased growth rates. However, the vegetative phase of aprf1 plants was prolonged and bolting was delayed, indicating an impairment in flowering under long days (LD). On the contrary, overexpression of APRF1 accelerates flowering. Consistent with the late flowering phenotype, the molecular data confirmed that FLC and SOC1 expression were significantly altered in the aprf1 mutants. Our data suggest that APRF1 acts upstream of FLC and promotes flowering under LD.

Auxin as an inducer of asymmetrical division generating the subsidiary cells in stomatal complexes of Zea mays.

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.

The interplay between ROS and tubulin cytoskeleton in plants.

Plants have to deal with reactive oxygen species (ROS) production, since it could potentially cause severe damages to different cellular components. On the other hand, ROS functioning as important second messengers are implicated in various developmental processes and are transiently produced during biotic or abiotic stresses. Furthermore, the microtubules (MTs) play a primary role in plant development and appear as potent players in sensing stressful situations and in the subsequent cellular responses. Emerging evidence suggests that ROS affect MTs in multiple ways. The cellular redox status seems to be tightly coupled with MTs. ROS signals regulate the organization of tubulin cytoskeleton and induce tubulin modifications. This review aims at summarizing the signaling mechanisms and the key operators orchestrating the crosstalk between ROS and tubulin cytoskeleton in plant cells. The contribution of several molecules, including microtubule associated proteins, oxidases, kinases, phospholipases, and transcription factors, is highlighted.

Plant cell division: ROS homeostasis is required.

Accumulated evidence indicates that ROS fluctuations play a critical role in cell division. Dividing plant cells rapidly respond to them. Experimental disturbance of ROS homeostasis affects: tubulin polymerization; PPB, mitotic spindle and phragmoplast assembly; nuclear envelope dynamics; chromosome separation and movement; cell plate formation. Dividing cells mainly accumulate at prophase and delay in passing through the successive cell division stages. Notably, many dividing root cells of the rhd2 Arabidopsis thaliana mutants, lacking the RHD2/AtRBOHC protein function, displayed aberrations, comparable to those induced by low ROS levels. Some protein molecules, playing key roles in signal transduction networks inducing ROS production, participate in cell division. NADPH oxidases and their regulators PLD, PI3K and ROP-GTPases, are involved in MT polymerization and organization. Cellular ROS oscillations function as messages rapidly transmitted through MAPK pathways inducing MAP activation, thus affecting MT dynamics and organization. RNS implication in cell division is also considered.

Disturbance of reactive oxygen species homeostasis induces atypical tubulin polymer formation and affects mitosis in root-tip cells of Triticum turgidum and Arabidopsis thaliana.

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.