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Although various studies on vibration control schemes exist, most of them focus on a one-time optimization of actuator layout. However, numerical results indicate significant drawbacks of fixed layouts on control performance, a factor rarely mentioned in the field of vibration control. This study aims to address this gap by proposing a novel rearrangement strategy that adaptively varies the actuator layout based on the vibration intensity pattern. The inspiration for this approach comes from the reorientation of cortical microtubules in plant cells, which adapt to stress patterns. The study first derives the definitions of vibration patterns and the index of intensity based on energy transfer. These definitions then guide the optimization of actuator layout throughout the control process. The study compares the fixed layout with the proposed adaptive varying layout, considering both full-state feedback and recognition feedback. The numerical results demonstrate that the proposed adaptive rearrangement of actuator layout significantly improves suppression performance, leading to higher utilization efficiency of control forces. Additionally, the presented strategy is tolerant to computation errors from recognition feedback, resulting in robust enhancements in vibration attenuation. Our study has introduced new possibilities for improving existing control schemes. • First-time practical dynamic rearrangement strategy adapts actuator layout based on system responses. • A bioinspired optimization framework aligns the actuator layout with a vibration intensity pattern in a fast-computing manner. • The strategy is compatible with both full-state feedback and recognition reconstructed from sensing signals. • Simulations show improved control performance with increased force utilization and error tolerance. [ABSTRACT FROM AUTHOR]

Summary: All living organisms are subjected to mechanical forces at all times. It has been reported that mechanics regulate many key cellular processes, including cell polarity establishment, cell division and gene expression, as a physical signal in both animal and plant development. Plant cells are exposed to several types of mechanical stresses, ranging from turgor‐driven tensile stresses, mechanical force modified by heterogeneous growth directions and rates between neighbouring cells, to forces from the environment such as wind and rain, for which they have developed adaptive mechanisms. Increasing evidence has revealed that mechanical stresses markedly influence the alignment of cortical microtubules (CMTs) in plant cells, among other effects. CMTs are able to reorient in response to mechanical stresses at both the single‐cell and tissue levels and always align with the maximal tensile stress direction. In this review, we discussed the known and potential molecules and pathways involved in the regulation of CMTs by mechanical stresses. We also summarized the available techniques that have allowed for mechanical perturbation. Finally, we highlighted several key questions remaining to be addressed in this emerging field. [ABSTRACT FROM AUTHOR]

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an 'ancestral' effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity. Author summary: Xanthomonas Outer Proteins (Xops) are type III effector proteins originating from bacterial plant pathogens of the Xanthomonas genus. Xanthomonas uses a needle-like structure to inject a cocktail of Xops directly into plant cells where they manipulate cellular processes to promote virulence. Previous studies of individual Xops have provided valuable insights into virulence strategies used by Xanthomonas, knowledge that can be exploited to fight plant disease. However, despite rapid progress in the field, there is much about effector activity we still do not understand. Our study focuses on the effector XopL, a protein with E3 ligase activity that is important for Xanthomonas virulence. In this study we expressed XopLs in leaves of the model plant N. benthamiana and found that XopLs from different Xanthomonas species differ in their subcellular localization. XopLs from closely related species associate with the microtubule cytoskeleton and disassemble it, whereas a XopL from a distantly related species did not. This prompted a comparative analysis of these proteins, which showed how microtubule binding is achieved and how it affects the plant response to XopL. [ABSTRACT FROM AUTHOR]

Although microtubules in plant cells have been extensively studied, the mechanisms that regulate the spatial organization of microtubules are poorly understood. We hypothesize that the interaction between microtubules and cytoplasmic flow plays an important role in the assembly and orientation of microtubules. To test this hypothesis, we developed a new computational modeling framework for microtubules based on theory and methods from the fluid–structure interaction. We employed the immersed boundary method to track the movement of microtubules in cytoplasmic flow. We also incorporated details of the encounter dynamics when two microtubules collide with each other. We verified our computational model through several numerical tests before applying it to the simulation of the microtubule–cytoplasm interaction in a growing plant cell. Our computational investigation demonstrated that microtubules are primarily oriented in the direction orthogonal to the axis of cell elongation. We validated the simulation results through a comparison with the measurement from laboratory experiments. We found that our computational model, with further calibration, was capable of generating microtubule orientation patterns that were qualitatively and quantitatively consistent with the experimental results. The computational model proposed in this study can be naturally extended to many other cellular systems that involve the interaction between microstructures and the intracellular fluid. [ABSTRACT FROM AUTHOR]

For the first time, electrochemical methods are utilized to study the response of tubulin monomers (extracted from plant source such as Green Peas: Arachis Hypogea) towards charge perturbations in the form of conductivity, conformational changes via self-assembly and adsorption on Au surface. The obtained dimerization and surface adsorption energetics of the tubulins from Cyclic Voltammetry agree well with the literature value of 6.9 and 14.9 kCal/mol for lateral and longitudinal bond formation energy respectively. In addition to the effects of charge perturbations on change in structure, ionic and electronic conductivity of tubulin with increasing load are investigated and found to be 1.25 Sm−1 and 2.89 mSm−1 respectively. The electronic conductivity is 1.93 times higher than the literature value of 1.5 mSm−1, demonstrating the fact that the microtubules (dimer of tubulins, MTs) from plant source can be used as a semiconductor electrode material in energy conversion and storage applications. Thus, motivated by the Monte Carlo simulation and electrochemical results the MTs extracted from plant source are used as cathode material for energy storage device such as Bio-battery and the Galvanostatic Charge/Discharge studies are carried out in coin cell configuration. The configuration of the bio-battery cell is as follows: Al/CB//PP-1M KCl//MTs/SS; where SS and Al are used as current collectors for cathode and anode respectively, Polypropylene (PP) membrane soaked in 1M KCl as electrolyte and Carbon Black (CB) is the anode material. Another configuration of the cell would be replacement of CB by biopolymer such as ethyl cellulose anode (Al/EC/PP-1M KCl/MTs/SS). [ABSTRACT FROM AUTHOR]

Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro. However, there was no investigation of TPs on microtubules in planta. Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules. [ABSTRACT FROM AUTHOR]

Specific populations of plant microtubules cooperate with the plasma membrane to sense and process abiotic stress signals, such as cold stress. The current study derived from the question, to what extent this perception system is active in biotic stress signalling. The experimental system consisted of grapevine cell lines, where microtubules or actin filaments are visualised by GFP, such that their response became visible in vivo. We used the bacterial elicitors harpin (inducing cell-death related defence), or flg22 (inducing basal immunity) in combination with modulators of membrane fluidity, or microtubules. We show that DMSO, a membrane rigidifier, can cause microtubule bundling and trigger defence responses, including activation of phytoalexin transcripts. However, DMSO inhibited the gene expression in response to harpin, while promoting the gene expression in response to flg22. Treatment with DMSO also rendered microtubules more persistent to harpin. Paradoxically, Benzylalcohol (BA), a membrane fluidiser, acted in the same way as DMSO. Neither GdCl3, nor diphenylene iodonium were able to block the inhibitory effect of membrane rigidification on harpin-induced gene expression. Treatment with taxol stabilised microtubule against harpin but amplified the response of PAL transcripts. Therefore, the data support implications of a model that deploys specific responses to pathogen-derived signals. [ABSTRACT FROM AUTHOR]

Tip-growth is a mode of polarized cell expansion where incorporation of new membrane and wall is stably restricted to a single, small domain of the cell surface resulting in the formation of a tubular projection that extends away from the body of the cell. The organization of the microtubule cytoskeleton is conserved among tip-growing cells of land plants: bundles of microtubules run longitudinally along the non-growing shank and a network of fine microtubules grow into the apical dome where growth occurs. Together, these microtubule networks control the stable positioning of the growth site at the cell surface. This conserved dynamic organization is required for the spatial stability of tip-growth, as demonstrated by the formation of sinuous tip-growing cells upon treatment with microtubule-stabilizing or microtubule-destabilizing drugs. Microtubule associated proteins (MAPs) that either stabilize or destabilize microtubule networks are required for the maintenance of stable tip-growth in root hairs of flowering plants. NIMA RELATED KINASE (NEK) is a MAP that destabilizes microtubule growing ends in the apical dome of tip-growing rhizoid cells in the liverwort Marchantia polymorpha. We hypothesized that both microtubule stabilizing and destabilizing MAPs are required for the maintenance of the stable tip-growth in liverworts. To identify genes encoding microtubule-stabilizing and microtubule-destabilizing activities we generated 120,000 UV-B mutagenized and 336,000 T-DNA transformed Marchantia polymorpha plants and screened for defective rhizoid phenotypes. We identified 119 mutants and retained 30 mutants in which the sinuous rhizoid phenotype was inherited. The 30 mutants were classified into at least 4 linkage groups. Characterisation of two of the linkage groups showed that MAP genes–WAVE DAMPENED2-LIKE (WDL) and NIMA-RELATED KINASE (NEK)–are required to stabilize the site of tip growth in elongating rhizoids. Furthermore, we show that MpWDL is required for the formation of a bundled array of parallel and longitudinally orientated microtubules in the non-growing shank of rhizoids where MpWDL-YFP localizes to microtubule bundles. We propose a model where the opposite functions of MpWDL and MpNEK on microtubule bundling are spatially separated and promote tip-growth spatial stability. Author summary: Plant cells control where they grow by adding membrane and cell wall material to a defined area of their surface. In particular, filamentous rooting cells develop the cellular projections essential to their function by restricting cell expansion to a stable domain of their surface. The spatial stability of this mechanism known as tip-growth defines the final shape of the cellular projections–straight projections form from stable tip-growth, while wavy or bifurcating projections form from unstable tip-growth. Microtubules are known to regulate tip-growth stability. Both microtubule stabilisation and destabilisation leads to unstable tip-growth. We have discovered two proteins that associate with microtubules, control their stability and are required for stabilizing tip-growth in the common liverwort. The first protein is known to destabilize microtubules in the tip of filamentous rooting cells of the common liverwort, and we found the second protein to stabilize, or bundle, microtubules in their shank. This is important because it is the first protein found to stabilize microtubules in the common liverwort and because it is the first time a protein stabilizing microtubules in rooting cells of plants is shown to localize separately from proteins that destabilizes microtubules. We propose that tip-growth stability requires the opposite functions of these two microtubule associated protein to be spatially separated. [ABSTRACT FROM AUTHOR]

The discovery and useful application of natural products can help improve human life. Chemicals that inhibit plant growth are broadly utilized as herbicides to control weeds. As various types of herbicides are required, the identification of compounds with novel modes of action is desirable. In the present study, we discovered a novel N-alkoxypyrrole compound, kumamonamide from Streptomyces werraensis MK493-CF1 and established a total synthesis procedure. Resulted in the bioactivity assays, we found that kumamonamic acid, a synthetic intermediate of kumamonamide, is a potential plant growth inhibitor. Further, we developed various derivatives of kumamonamic acid, including a kumamonamic acid nonyloxy derivative (KAND), which displayed high herbicidal activity without adverse effects on HeLa cell growth. We also detected that kumamonamic acid derivatives disturb plant microtubules; and additionally, that KAND affected actin filaments and induced cell death. These multifaceted effects differ from those of known microtubule inhibitors, suggesting a novel mode of action of kumamonamic acid, which represents an important lead for the development of new herbicides. [ABSTRACT FROM AUTHOR]

Plants can use volatiles for remote suppression of competitors. Mints produce essential oils, which are known to affect the growth of other plants. We used a comparative approach to identify allelopathic compounds from different Mints (genus Mentha , but also including Cat Mint, Nepeta cataria , and Corean Mint, Agastache rugosa , belonging to sisters clades within the Mentheae) using the standard cress germination assay as readout. To understand the mechanism behind this allelopathic effect, we investigated the response of tobacco BY-2 cell lines, expressing GFP-tagged markers for microtubules and actin filaments to these essential oils. Based on the comparison between bioactivity and chemical components, we identified menthone as prime candidate for the allelopathic effect, and confirmed this bioactivity targeted to microtubules experimentally in both, plant cells (tobaccoBY-2), and seedlings (Arabidopsis thaliana). We could show that menthone disrupted microtubules and induced mortality linked with a rapid permeabilization (less than 15 min) of the plasma membrane. This mortality was elevated in a tubulin marker line, where microtubules are mildly stabilized. Our study paves the way for the development of novel bioherbicides that would be environmentally friendly. [ABSTRACT FROM AUTHOR]

In plants, microtubule and actin cytoskeletons are involved in key processes including cell division, cell expansion, growth and development, biotic and abiotic stress, tropisms, hormonal signalling as well as cytoplasmic streaming in growing pollen tubes. Kinesin enzymes have a highly conserved motor domain for binding microtubule cytoskeleton assisting these motors to organise their own tracks, the microtubules by using chemical energy of ATP hydrolysis. In addition to this conserved binding site, kinesins possess non-conserved variable domains mediating structural and functional interaction of microtubules with other cell structures to perform various cellular jobs such as chromosome segregation, spindle formation and elongation, transport of organelles as well as microtubules-actins cross linking and microtubules sliding. Therefore, how the non-motor variable regions specify the kinesin function is of fundamental importance for all eukaryotic cells. Kinesins are classified into ~17 known families and some ungrouped orphans, of which ~13 families have been recognised in plants. Kinesin-14 family consisted of plant specific microtubules minus end-directed motors, are much diverse and unique to plants in the sense that they substitute the functions of animal dynein. In this review, we explore the functions of plant kinesins, especially from non-motor domains viewpoint, focussing mainly on recent work on the origin and functional diversity of motors that drive microtubule minus-end trafficking events. Correct positioning of nuclei and transport of cellular organelles and vesicles are essential for cell division and movement, proper growth and development of an organism. To perform these functions, organisms use cytoskeletal tracks on which molecular motors (kinesin, dynein and myosin) drive and transport their specific attached cargoes. In this review, we explore the functions of plant kinesin family motors with special focus on the similarities and dissimilarities existed in different plant species with respect to their specific reported cargoes. [ABSTRACT FROM AUTHOR]

The plant microtubules form unique arrays using acentrosomal microtubule nucleation pathways, yet utilizing evolutionary conserved centrosomal proteins. In cytokinesis, a multi-component cytoskeletal apparatus, the phragmoplast mediates the biosynthesis of the new cell plate by dynamic centrifugal expansion, a process that demands exquisite coordination of microtubule turnover and endomembrane trafficking. At the same time, the phragmoplast is guided to meet with the parental wall at a cortical site that is predefined before mitotic entry and transiently marked by the preprophase band of microtubules. The cortical division zone maintains positional information of the selected division plane for the entire duration of cell division and for the guidance of the phragmoplast during cytokinesis. Its establishment is an essential requirement for normal plant organogenesis, due to the confinement of cells by rigid cell walls. [ABSTRACT FROM AUTHOR]

Microtubule-dependent microtubule nucleation occurs on the lateral surface of pre-existing microtubules and provides a highly efficient means of amplifying their populations and reorganizing their architectures. The γ‑tubulin ring complex serves as the template to initiate nascent microtubule polymerization. Augmin, a hetero-octameric protein complex, acts as a recruiting factor to target the γ‑tubulin ring complex to pre-existing microtubules and trigger new microtubule growth. Although microtubule-dependent microtubule nucleation has been extensively studied in both animal and plant cells, it remains unclear how the augmin complex assembles in plant cells, especially in cell-cycle-specific and cell-type-specific manners, and how its spatial structure orchestrates the nucleation geometry. In this review, we summarize the advances in knowledge of augmin-dependent microtubule nucleation and the regulation of its geometry, and highlight recent findings and emerging questions concerning the role of the augmin complex in establishing microtubule arrays and the cell-cycle-specific composition of augmin in plant cells. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Lactic acid production with free and immobilized L. paracasei was studied. • Three lignocellulosic materials were used as supports for cell immobilization. • Physical characterization of support materials was performed. • Strong cell attachment enabled reuse of immobilized biomass in five batch cycles. • The highest LA productivity was achieved in sugar beet pulp immobilized system. Abstract In this study, the production of lactic acid (LA) on sugar beet molasses enriched potato stillage by immobilized Lactobacillus paracasei NRRL B-4564 was examined. Three agro-industrial materials, such as sunflower seed hull (SSH), brewers' spent grain (BSG), and sugar beet pulp (SBP) were studied as carriers for cell immobilization. The carriers were physically characterized in terms of water adsorption index (WAI), critical humidity point (CHP) and porosity. Further, the stability and efficiency of the immobilized biocatalysts were evaluated in repeated batch fermentation of molasses enriched potato stillage and compared with free cell system. A strong cell attachment onto agro-industrial supports allowed easy separation from the fermentation media and efficient biocatalyst reuse in five successive batch cycles. The highest cell number attached on the support surface during the fermentation was detected for material with higher WAI and lower CHP. Porosimetry measurements showed that the attachment of L. paracasei cells to support materials and overall LA productivities achieved with different immobilized biocatalysts were not determined by the support porosity and surface morphology, but with the material characteristics such as electrostatic charge, chemical composition and hydrophilicity. LA productivity of 1.48 g/L h, maximal LA concentration of 80.10 g/L and average yield coefficient of 0.97 g/g were achieved in fermentation of molasses enriched potato stillage using SBP as a support material, followed by BSG and SSH. The studied approach showed to be an interesting alternative strategy for increasing the LA productivities on low-cost and abundant substrate. [ABSTRACT FROM AUTHOR]

The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events. [ABSTRACT FROM AUTHOR]

Many cell functions rely on the ability of microtubules to self-organize as complex networks. In plants, cortical microtubules are essential to determine cell shape as they guide the deposition of cellulose microfibrils, and thus control mechanical anisotropy of the cell wall. Here we analyze how, in turn, cell shape may influence microtubule behavior. Buidling upon previous models that confined microtubules to the cell surface, we introduce an agent model of microtubules enclosed in a three-dimensional volume. We show that the microtubule network has spontaneous aligned configurations that could explain many experimental observations without resorting to specific regulation. In particular, we find that the preferred cortical localization of microtubules emerges from directional persistence of the microtubules, and their interactions with each other and with the stiff wall. We also identify microtubule parameters that seem relatively insensitive to cell shape, such as length or number. In contrast, microtubule array anisotropy depends on local curvature of the cell surface and global orientation follows robustly the longest axis of the cell. Lastly, we find that geometric cues may be overcome, as network is capable of reorienting toward weak external directional cues. Altogether our simulations show that the microtubule network is a good transducer of weak external polarity, while at the same time, easily reaching stable global configurations. [ABSTRACT FROM AUTHOR]

Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes. [ABSTRACT FROM AUTHOR]

Plant microtubules (MTs) are nucleated from the γ-tubulin-containing ring complex (γTuRC). In cortical MT arrays of interphase plant cells, γTuRC is preferentially recruited to the lattice of preexisting MTs, where it initiates MT nucleation in either a branch- or bundle-forming manner, or dissociates without mediating nucleation. In this study, we analyzed how γTuRCs influence MT nucleation and dynamics in cotyledon pavement cells of Arabidopsis thaliana. We found that γTuRC nucleated MTs at angles of ~40° toward the plus-ends of existing MTs, or in predominantly antiparallel bundles. A small fraction of γTuRCs was motile and tracked MT ends. When γTuRCs decorated the depolymerizing MT end, they reduced the depolymerization rate. Non-nucleating γTuRCs associated with the MT lattice promoted MT regrowth after a depolymerization phase. These results suggest that γTuRCs not only nucleate MT growth but also regulate MT dynamics by stabilizing MT ends. On rare occasions, a non-MT-associated γTuRC was pushed in the direction of the MT minus-end, while nucleating a new MT, suggesting that the polymerizing plus-end is anchored to the plasma membrane. [ABSTRACT FROM AUTHOR]

Plant morphogenesis relies on the accurate positioning of the partition (cell plate) between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which consists of microtubules, actin filaments, membrane compartments and associated proteins. The phragmoplast forms between daughter nuclei during the transition from anaphase to telophase. As cells are commonly larger than the originally formed phragmoplast, the construction of the cell plate requires phragmoplast expansion. This expansion depends on microtubule polymerization at the phragmoplast forefront (leading zone) and loss at the back (lagging zone). Leading and lagging zones sandwich the 'transition' zone. A population of stable microtubules in the transition zone facilitates transport of building materials to the midzone where the cell plate assembly takes place. Whereas microtubules undergo dynamic instability in all zones, the overall balance appears to be shifted towards depolymerization in the lagging zone. Polymerization of microtubules behind the lagging zone has not been reported to date, suggesting thatmicrotubule loss there is irreversible. In this Review, we discuss: (1) the regulation of microtubule dynamics in the phragmoplast zones during expansion; (2) mechanisms of the midzone establishment and initiation of cell plate biogenesis; and (3) signaling in the phragmoplast. [ABSTRACT FROM AUTHOR]

Plant cortical microtubules have crucial roles in cell wall development. Cortical microtubules are tightly anchored to the plasma membrane in a highly ordered array, which directs the deposition of cellulose microfibrils by guiding the movement of the cellulose synthase complex. Cortical microtubules also interact with several endomembrane systems to regulate cell wall development and other cellular events. Recent studies have identified new factors that mediate interactions between cortical microtubules and endomembrane systems including the plasma membrane, endosome, exocytic vesicles, and endoplasmic reticulum. These studies revealed that cortical microtubule-membrane interactions are highly dynamic, with specialized roles in developmental and environmental signaling pathways. A recent reconstructive study identified a novel function of the cortical microtubule-plasma membrane interaction, which acts as a lateral fence that defines plasma membrane domains. This review summarizes recent advances in our understanding of the mechanisms and functions of cortical microtubule-membrane interactions. [ABSTRACT FROM AUTHOR]

The cell wall is a complex polysaccharide network that provides stability and protection to the plant and is one of the first layers of biotic and abiotic stimuli perception. A controlled remodeling of the primary cell wall is essential for the plant to adapt its growth to environmental stresses. Cellulose, the main component of plant cell walls is synthesized by plasma membrane-localized cellulose synthases moving along cortical microtubule tracks. Recent advancements demonstrate a tight regulation of cellulose synthesis at the primary cell wall by phytohormone networks. Stress-induced perturbations at the cell wall that modify cellulose synthesis and microtubule arrangement activate similar phytohormone-based stress response pathways. The integration of stress perception at the primary cell wall and downstream responses are likely to be tightly regulated by phytohormone signaling pathways in the context of cellulose synthesis and microtubule arrangement. [ABSTRACT FROM AUTHOR]

Proper patterning of the cell wall is essential for plant cell development. Cortical microtubule arrays direct the deposition patterns of cell walls at the plasma membrane. However, the precise mechanism underlying cortical microtubule organization is not well understood. Here, we show that a microtubule-associated protein, CORD1 (CORTICAL MICROTUBULE DISORDERING1), is required for the pitted secondary cell wall pattern of metaxylem vessels in Arabidopsis thaliana. Loss of CORD1 and its paralog, CORD2 , led to the formation of irregular secondary cell walls with small pits in metaxylem vessels, while overexpressing CORD1 led to the formation of abnormally enlarged secondary cell wall pits. Ectopic expression of CORD1 disturbed the parallel cortical microtubule array by promoting the detachment of microtubules from the plasma membrane. A reconstructive approach revealed that CORD1-induced disorganization of cortical microtubules impairs the boundaries of plasma membrane domains of active ROP11 GTPase, which govern pit formation. Our data suggest that CORD1 promotes cortical microtubule disorganization to regulate secondary cell wall pit formation. The Arabidopsis genome has six CORD1 paralogs that are expressed in various tissues during plant development, suggesting they are important for regulating cortical microtubules during plant development. [ABSTRACT FROM AUTHOR]

Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1ΔARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1ΔARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression. [ABSTRACT FROM AUTHOR]

The reorganization of microtubules induced by salt stress is required for Arabidopsis survival under high salinity conditions. RIC1 is an effector of Rho-related GTPase from plants (ROPs) and a known microtubule-associated protein. In this study, we demonstrated that RIC1 expression decreased with long-term NaCl treatment, and ric1-1 seedlings exhibited a higher survival rate under salt stress. We found that RIC1 reduced the frequency of microtubule transition from shortening to growing status and knockout of RIC1 improved the reassembly of depolymerized microtubules caused by either oryzalin treatment or salt stress. Further investigation showed that constitutively active ROP2 promoted the reassembly of microtubules and the survival of seedlings under salt stress. A rop2-1 ric1-1 double mutant rescued the salt-sensitive phenotype of rop2-1, indicating that ROP2 functions in salt tolerance through RIC1. Although ROP2 did not regulate RIC1 expression upon salt stress, a quick but mild increase of ROP2 activity was induced, led to reduction of RIC1 on microtubules. Collectively, our study reveals an ROP2-RIC1 pathway that fine-tunes microtubule dynamics in response to salt stress in Arabidopsis. This finding not only reveals a new regulatory mechanism for microtubule reorganization under salt stress but also the importance of ROP signalling for salinity tolerance. [ABSTRACT FROM AUTHOR]

Minus end-directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end-directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plantspecific class VI kinesin-14, KCBP, is required for minus end-directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end-directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end-directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end-directed transporters in land plants. [ABSTRACT FROM AUTHOR]

Katanin is the only microtubule severing protein identified in plants so far. Previous studies have documented its role in regulating cortical microtubule organization during cell growth and morphogenesis. Although, some cell division defects are reported in KATANIN mutants, it is not clear whether or how katanin activity may affect microtubule dynamics in interphase cells, as well as the progression of mitosis and cytokinesis and the orientation of cell division plane (CDP). For this reason, we characterized microtubule organization and dynamics in growing and dividing cotyledon cells of Arabidopsis ktn1-2 mutant devoid of KATANIN 1 activity. In interphase epidermal cells of ktn1-2 cortical microtubules exhibited aberrant and largely isotropic organization, reduced bundling and showed excessive branchedmicrotubule formation. End-wisemicrotubule dynamics were not much affected, although a significantly slower rate of microtubule growth was measured in the ktn1-2 mutant where microtubule severing was completely abolished. KATANIN 1 depletion also brought about significant changes in preprophase microtubule band (PPB) organization and dynamics. In this case, many PPBs exhibited unisided organization and splayed appearance while in most cases they were broader than those of wild type cells. By recording PPB maturation, it was observed that PPBs in the mutant narrowed at a much slower pace compared to those in Col-0. The form of the mitotic spindle and the phragmoplast was not much affected in ktn1-2, however, the dynamics of both processes showed significant differences compared to wild type. In general, both mitosis and cytokinesis were considerably delayed in the mutant. Additionally, the mitotic spindle and the phragmoplast exhibited extensive rotational motions with the equatorial plane of the spindle being essentially uncoupled from the division plane set by the PPB. However, at the onset of its formation the phragmoplast undergoes rotationalmotion rectifying the expansion of the cell plate tomatch the original cell division plane. Conclusively, KATANIN 1 contributes to microtubule dynamics during interphase, regulates PPB formation and maturation and is involved in the positioning of the mitotic spindle and the phragmoplast. [ABSTRACT FROM AUTHOR]

In the article, the author discusses the benefits of mentoring undergraduate research students to enable them to achieve a degree of independence with the scientific inquiry process. He cites his own experiences with mentors Doctors Regina McClinton and Aikseng Ooi during his project to clone and sequence a gene and microtubules of the Arabidopsis family of plants. Also mentioned are the skills he learned like critical thinking, perseverance, and imagination.

Background: Detailed descriptions of the early development of parasitic nematodes are seldom available. The embryonic development of the plant-parasitic nematode Meloidogyne incognita was studied, focusing on the early events. Results: A fixed pattern of repeated cell cleavages was observed, resulting in the appearance of the six founder cells 3 days after the first cell division. Gastrulation, characterized by the translocation of cells from the ventral side to the center of the embryo, was seen 1 day later. Approximately 10 days after the first cell division a rapidly elongating two-fold stage was reached. The fully developed second stage juvenile hatched approximately 21 days after the first cell division. Conclusions: When compared to the development of the free-living nematode Caenorhabditis elegans, the development of M. incognita occurs approximately 35 times more slowly. Furthermore, M. incognita differs from C. elegans in the order of cell divisions, and the early cleavage patterns of the germ line cells. However, cytoplasmic ruffling and nuclear migration prior to the first cell division as well as the localization of microtubules are similar between C. elegans and M. incognita. [ABSTRACT FROM AUTHOR]

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Wholetranscriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection. [ABSTRACT FROM AUTHOR]

The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane., To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed., Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation., The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division. [ABSTRACT FROM AUTHOR]

The cytoskeleton in pollen tubes has been intensively studied, because of its abundance and prominent roles and because the pollen tube is an excellent experimental system for cell biological studies. Pollen actin microfilaments (MFs) exist as multiple distinct populations, each participating in a specific cellular trafficking or organization process. Consequently, MFs are essential for pollen tube growth and are tightly regulated in response to various signals. Pollen microtubules (MTs) are non-essential and less characterized, but recent studies have implicated MTs in vesicle trafficking and cell wall construction in pollen tubes. This review summarizes recent advances in understanding the organization and regulation of both MFs and MTs and discusses their roles in cellular trafficking and the modulation of pollen-tube tip growth. [ABSTRACT FROM AUTHOR]

Plants produce enormous forms of the microtubule (MT)-based motor kinesins that have been inspiring plant cell biologists to uncover their functions in relation to plant growth and development. Subcellular localization of kinesin proteins detected through live-cell imaging or immunofluorescence microscopy has provided great insights into the functions of these motors. Dozens of mitotic kinesins exhibit particularly splendid localization patterns from chromosomes and kinetochores to MT arrays like the preprophase band, spindle poles, the spindle midzone, phragmoplast distal ends, and the phragmoplast midzone. Different subcellular localizations indicate distinct functions of these motors that are yet to be characterized. The localization difference between plant kinesins and their animal counterparts implies mechanistic differences in mitosis and cytokinesis between the two kingdoms. When many forms of kinesins are present simultaneously, it becomes critical that their motility is differentially regulated with spatial and temporal precision. Insights into regulatory mechanisms of motors can often be brought about by in vitro single-molecule biophysical studies. Significant advances are expected in this area in the coming years owing to rapid technological advances that are being brought to various model plants. [ABSTRACT FROM AUTHOR]

Plant cortical microtubules can form ordered arrays through interactions among themselves. When an incident microtubule collides with a barrier microtubule it may entrain if below a certain angle. Else it undergoes collision induced catastrophe (CIC) or crosses over the barrier microtubule. It has been proposed that katanin is necessary to create order by severing these crossover sites. The authors present a three-state computational model using Arabidopsis thaliana data to show how spontaneous catastrophe, the probability of CIC versus crossover, and katanin-mediated severing at the crossover sites affect microtubule ordering. The results of the systematic simulations show that (1), the microtubule order is more sensitive to the catastrophe rate than the rescue rate; (2), at 21°C, peak order is observed at 0.3 CIC and order decreases as CIC increases; and (3) at 0.2 CIC, katanin severing acting uniformly at all crossover sites is able to create order within a biologically reasonable time frame, but at lower CICs this becomes unrealistically fast. This would imply that at lower CIC levels preferential crossover site targeting and severing activity regulators would be required for katanin to bring about order. [ABSTRACT FROM AUTHOR]

Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues during regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis. [ABSTRACT FROM AUTHOR]

Jatropha curcas is an oil-rich Euphorbiaceae seed species renowned for its apparent tolerance to environmental stresses. It is considered a promising source of renewable feedstock for biodiesel production in the Brazilian semiarid region where crop establishment requires a better understanding of the mechanisms leading to proper seed and plant behavior under water restrictive conditions. This study describes physiological and cytological profiles of J. curcas seeds imbibed in water restriction conditions by means of osmotic stress or osmoconditioning. Seeds were characterized by size, weight, moisture content and dry mass, germinability, and cell cycle activation by means of tubulin and microtubule cytoskeleton accumulation. Osmoconditioning at -0.8 MPa did not induce priming effects as it did not improve the physiological quality of the seed lots. Western blotting and immunocytochemical analysis revealed an increasing accumulation of tubulin and microtubule cytoskeleton in seeds imbibed in water for 48h onwards, culminating in the onset of mitotic configurations after germination. Only cortical microtubules were observed during seed osmoconditioning, whereas mitotic microtubules only occurred after reimbibition of osmoconditioned seeds in water and subsequent germination. [ABSTRACT FROM AUTHOR]

Plant microtubules (MTs), in addition to their role in cell division and cell expansion, respond to various stress signals. To understand the biological function of this early response requires non-destructive strategies for visualization in cellular models that are highly responsive to stress signals. We have therefore generated a transgenic tubulin marker line for a cell line from the grapevine Vitis rupestris that readily responds to stress factors of defense-related and abiotic stresses based on a fusion of the green fluorescent protein with Arabidopsis β-tubulin 6. By a combination of spinning-disk confocal microscopy with quantitative image analysis, we could detect early and specific responses of MTs to defense-related and abiotic stress factors in vivo . We observed that Harpin Z (HrpZ), a bacterial elicitor that can trigger programmed cell death, rapidly eliminated radial MTs, followed by a slower depletion of the cortical array. Jasmonic acid (JA), in contrast, induced bundling of cortical MTs. Auxin reduced the thickness of cortical MTs. This effect followed a characteristic bell-shaped dose-dependency and could revert JA-induced bundling. Impeded cell expansion as a consequence of stress treatment or superoptimal auxin was linked with the appearance of intranuclear tubulin speckles. The early and stimulus-specific responses of MTs are discussed with respect to a function in processing or decoding of stress signals. [ABSTRACT FROM AUTHOR]

In plants, actin filaments have an important role in organelle movement and cytoplasmic streaming. Otherwise microtubules (MTs) have a role in restricting organelles to specific areas of the cell and in maintaining organelle morphology. In somatic plant cells, MTs also participate in cell division and morphogenesis, allowing cells to take their definitive shape in order to perform specific functions. In the latter case, MTs influence assembly of the cell wall, controlling the delivery of enzymes involved in cellulose synthesis and of wall modulation material to the proper sites. In angiosperm pollen tubes, organelle movement is generally attributed to the acto-myosin system, the main role of which is in distributing organelles in the cytoplasm and in carrying secretory vesicles to the apex for polarized growth. Recent data on membrane trafficking suggests a role of MTs in fine delivery and repositioning of vesicles to sustain pollen tube growth. This review examines the role of MTs in secretion and endocytosis, highlighting new research cues regarding cell wall construction and pollen tube-pistil crosstalk, that help unravel the role of MTs in polarized growth. [ABSTRACT FROM AUTHOR]

The cytoskeleton plays a central part in spatial organization of the plant cytoplasm, including the endomebrane system. However, the mechanisms involved are so far only partially understood. Formins (FH2 proteins), a family of evolutionarily conserved proteins sharing the FH2 domain whose dimer can nucleate actin, mediate the co-ordination between actin and microtubule cytoskeletons in multiple eukaryotic lineages including plants. Moreover, some plant formins contain transmembrane domains and participate in anchoring cytoskeletal structures to the plasmalemma, and possibly to other membranes. Direct or indirect membrane association is well documented even for some fungal and metazoan formins lacking membrane insertion motifs, and FH2 proteins have been shown to associate with endomembranes and modulate their dynamics in both fungi and metazoans. Here we summarize the available evidence suggesting that formins participate in membrane trafficking and endomembrane, especially ER, organization also in plants. We propose that, despite some methodological pitfalls inherent to in vivo studies based on (over)expression of truncated and/or tagged proteins, formins are beginning to emerge as candidates for the so far somewhat elusive link between the plant cytoskeleton and the endomembrane system. [ABSTRACT FROM AUTHOR]

Within living cells, the transport of cargo is accomplished by groups of molecular motors. Such collective transport could utilize mechanisms which emerge from inter-motor interactions in ways that are yet to be fully understood. Here we combined experimental measurements of two-kinesin transport with a theoretical framework to investigate the functional ramifications of inter-motor interactions on individual motor function and collective cargo transport. In contrast to kinesin's low sidestepping frequency when present as a single motor, with exactly two kinesins per cargo, we observed substantial motion perpendicular to the microtubule. Our model captures a surface-associated mode of kinesin, which is only accessible via inter-motor interference in groups, in which kinesin diffuses along the microtubule surface and rapidly ''hops'' between protofilaments without dissociating from the microtubule. Critically, each kinesin transitions dynamically between the active stepping mode and this weak surface-associated mode enhancing local exploration of the microtubule surface, possibly enabling cellular cargos to overcome macromolecular crowding and to navigate obstacles along microtubule tracks without sacrificing overall travel distance. [ABSTRACT FROM AUTHOR]

Throughout the wheat-growing regions of Australia, chilling temperatures below 2 °C occur periodically on consecutive nights during the period of floral development in spring wheat ( T riticum aestivum L.). In this study, wheat plants showed significant reductions in fertility when exposed to prolonged chilling temperatures in controlled environment experiments. Among the cultivars tested, the Australian cultivars Kite and Hartog had among the lowest levels of seed set due to chilling and their responses were investigated further. The developmental stage at exposure, the chilling temperature and length of exposure all influenced the level of sterility. The early period of booting, and specifically the +4 cm auricle distance class, was the most sensitive and corresponded to meiosis within the anthers. The response of microtubules to chilling during meiosis in Hartog was monitored, but there was little difference between chilled and control plants. Other abnormalities, such as plasmolysis and cytomixis increased in frequency, were associated with death of developing pollen cells, and could contribute to loss of fertility. The potential for an above-zero chilling sensitivity in Australian spring wheat varieties could have implications for exploring the tolerance of wheat flower development to chilling and freezing conditions in the field. [ABSTRACT FROM AUTHOR]

The article discusses research which investigates the expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analyzes the microtubule assembly in the presence of taxol. Topics discussed include isolation of taxol from the yew Taxus brevifolia, cloning of the complimentary DNA (cDNA)for both tubulins from Taxus cuspidata and the human embryonic kidney cell line HEK293T, and concludes less senitivity of microtubule from cuspidata to taxol than that from HEK293T.

Microtubules are oriented into transverse arrays in response to growth-promoting hormones. This reorientation process does not involve changes in the proportion of different nucleation modes at the periclinal cell surface.Aligned microtubule arrays spatially organize cell division, trafficking, and determine the direction of cell expansion in plant cells. In response to changes in environmental and developmental signals, cells reorganize their microtubule arrays into new configurations. Here, we tested the role of microtubule nucleation during hormone-induced microtubule array reorientation. We have found that in the process of microtubule array reorientation the ratios between branching, parallel, and de-novo nucleations remained constant, suggesting that the microtubule reorientation mechanism does not involve changes in nucleation modes. In the ton2/fass mutant, which has reduced microtubule branching nucleation frequency and decreased nucleation activity of the γ-tubulin complexes, microtubule arrays were able to reorient. Presented data suggest that reorientation of microtubules into transverse arrays in response to hormones does not involve changes in microtubule nucleation at the periclinal cell surface [ABSTRACT FROM AUTHOR]

Microtubules are essential for various cellular activities and β-tubulins are the target of benzimidazole fungicides. However, the evolution and molecular mechanisms driving functional diversification in fungal tubulins are not clear. In this study, we systematically identified tubulin genes from 59 representative fungi across the fungal kingdom. Phylogenetic analysis showed that α-/β-tubulin genes underwent multiple independent duplications and losses in different fungal lineages and formed distinct paralogous/ orthologous clades. The last common ancestor of basidiomycetes and ascomycetes likely possessed two paralogs of a-tubulin (a1/a2) and β-tubulin (β1/β2) genes but α2-tubulin genes were lost in basidiomycetes and β2-tubulin genes were lost in most ascomycetes. Molecular evolutionary analysis indicated that α1, α2, and β2-tubulins have been under strong divergent selection and adaptive positive selection. Many positively selected sites are at or adjacent to important functional sites and likely contribute to functional diversification. We further experimentally confirmed functional divergence of two β-tubulins in Fusarium and identified type II variations in FgTub2 responsible for function shifts. In this study, we also identified δ-/ϵ-/η-tubulins in Chytridiomycetes. Overall, our results illustrated that different evolutionary mechanisms drive functional diversification of α-/β-tubulin genes in different fungal lineages, and residues under positive selection could provide targets for further experimental study. [ABSTRACT FROM AUTHOR]

Microtubules are one of the three major cytoskeletal components in eukaryotic cells. Heterodimers composed of GTP-bound α- and β-tubulin molecules polymerize to form microtubule protofilaments, which associate laterally to form a hollow microtubule.Tubulin has GTPase activity and the GTP molecules associated with β-tubulin molecules are hydrolyzed shortly after being incorporated into the polymerizing microtubules. GTP hydrolysis alters the conformation of the tubulin molecules and drives the dynamic behavior of microtubules. Periods of rapid microtubule polymerization alternate with periods of shrinkage in a process known as dynamic instability. In plants, dynamic instability plays a key role in determining the organization of microtubules into arrays, and these arrays vary throughout the cell cycle. In this review, we describe the mechanisms that regulate microtubule dynamics and underlie dynamic instability, and discuss howdynamic instability may shape microtubule organization in plant cells. [ABSTRACT FROM AUTHOR]

The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth. [ABSTRACT FROM PUBLISHER]

The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER). Thus, to probe the significance of dynamic MT behavior in the coordination of MT-network-associated functions related to TMV infection and, thus, in the formation and transport of RNA complexes in the cytoplasm, we performed an evolution experiment with TMV in Arabidopsis thaliana tor1/spr2 and tor2 mutants with specific defects in MT dynamics and asked whether TMV is sensitive to these changes. We show that the altered cytoskeleton induced genetic changes in TMV that were correlated with efficient spread of infection in the mutant hosts. These observations demonstrate a role of dynamic MT rearrangements and of the MT-associated protein TORTIFOLIA1/SPIRAL2 in cellular functions related to virus spread and indicate that MT dynamics and MT-associated proteins represent constraints for virus evolution and adaptation. The results highlight the importance of the dynamic plasticity of the MT network in directing cytoplasmic functions in macromolecular assembly and trafficking and illustrate the value of experimental virus evolution for addressing the cellular functions of dynamic, long-range order systems in multicellular organisms. [ABSTRACT FROM AUTHOR]

Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability. [ABSTRACT FROM AUTHOR]