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  1. Abstract

    Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP–RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

     
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  2. Introduction

    During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles.

    Methods

    Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells.

    Results

    The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects inArabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins.

    Discussion

    Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

     
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  3. Abstract Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a “cell plate” partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2–6 pN/nm, an osmotic pressure difference of 2–10 kPa, and spontaneous curvature between 0 and 0.04 nm−1. With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model’s concept, suggesting that it is one of the factors involved in establishing the spreading force. 
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  4. null (Ed.)
    If you live in an apartment or a house, you will notice that your home has different rooms separated by walls. A plant is just like your home, except there are many small rooms, called cells. Plant cells, like rooms, are also separated by cell walls. Cell walls are unique and are not found in animal cells. In a building, if you want to turn one large room into two small rooms, you build a new wall to divide it. This is similar to how a plant cell divides into two cells during cell division. To build a wall in a building, you need to employ construction workers, design the building plan, buy building materials, and finally assembly the wall. How does the plant cell take care of these different jobs? This article explains how the cell wall is built in a plant cell during cell division. 
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  5. null (Ed.)
    Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose, a β-1,3 glucan accumulates at later stages of cell plate development presumably to stabilize this delicate membrane network during expansion. Cytokinetic callose is considered specific to multicellular plant species, as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, P. margaritaceum. Callose deposition at the division plane of P. margaritaceum showed distinct, spatiotemporal patterns likely representing distinct roles of this polymer in cytokinesis. Pharmacological inhibition by Endosidin 7 resulted in cytokinesis defects, consistent with the essential role for this polymer in P. margaritaceum cell division. Cell wall deposition at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly in P. margaritaceum. The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis in P. margaritaceum. The evolutionary implications of cytokinetic callose in this unicellular Zygnematopycean alga is discussed in the context of the conquest of land by plants. 
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  6. Baskin, Tobias Isaac (Ed.)
    In plant cytokinesis, de novo formation of a cell plate evolving into the new cell wall partitions the cytoplasm of the dividing cell. In our earlier chemical genomics studies, we identified and characterized the small molecule endosidin-7, that specifically inhibits callose deposition at the cell plate, arresting late-stage cytokinesis in arabidopsis. Endosidin-7 has emerged as a very valuable tool for dissecting this essential plant process. To gain insights regarding its mode of action and the effects of cytokinesis inhibition on the overall plant response, we investigated the effect of endosidin-7 through a nuclear magnetic resonance spectroscopy (NMR) metabolomics approach. In this case study, metabolomics profiles of arabidopsis leaf and root tissues were analyzed at different growth stages and endosidin-7 exposure levels. The results show leaf and root-specific metabolic profile changes and the effects of endosidin-7 treatment on these metabolomes. Statistical analyses indicated that the effect of endosidin-7 treatment was more significant than the developmental impact. The endosidin-7 induced metabolic profiles suggest compensations for cytokinesis inhibition in central metabolism pathways. This study further shows that long-term treatment of endosidin-7 profoundly changes, likely via alteration of hormonal regulation, the primary metabolism of arabidopsis seedlings. Hormonal pathway-changes are likely reflecting the plant’s responses, compensating for the arrested cell division, which in turn are leading to global metabolite modulation. The presented NMR spectral data are made available through the Metabolomics Workbench, providing a reference resource for the scientific community. 
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