Comprehensive and quantitative analysis of intracellular structure polarization at the apical-basal axis in elongating Arabidopsis zygotes.

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

Novel inhibitors of microtubule organization and phragmoplast formation in diverse plant species.

Cell division is essential for development and involves spindle assembly, chromosome separation, and cytokinesis. In plants, the genetic tools for controlling the events in cell division at the desired time are limited and ineffective owing to high redundancy and lethality. Therefore, we screened cell division-affecting compounds in Arabidopsis thaliana zygotes, whose cell division is traceable without time-lapse observations. We then determined the target events of the identified compounds using live-cell imaging of tobacco BY-2 cells. Subsequently, we isolated two compounds, PD-180970 and PP2, neither of which caused lethal damage. PD-180970 disrupted microtubule (MT) organization and, thus, nuclear separation, and PP2 blocked phragmoplast formation and impaired cytokinesis. Phosphoproteomic analysis showed that these compounds reduced the phosphorylation of diverse proteins, including MT-associated proteins (MAP70) and class II Kinesin-12. Moreover, these compounds were effective in multiple plant species, such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes conserved across diverse plant species.

Dynamic Rearrangement and Directional Migration of Tubular Vacuoles are Required for the Asymmetric Division of the Arabidopsis Zygote.

In most flowering plants, the asymmetric cell division of zygotes is the initial step that establishes the apical-basal axis. In the Arabidopsis zygote, vacuolar accumulation at the basal cell end is crucial to ensure zygotic division asymmetry. Despite the importance, it was unclear whether this polar vacuolar distribution was achieved by predominant biogenesis at the basal region or by directional movement after biogenesis. Here, we found that apical and basal vacuolar contents are dynamically exchanged via a tubular vacuolar network and the vacuoles gradually migrate toward the basal end. The mutant of a vacuolar membrane protein, SHOOT GRAVITROPISM2 (SGR2), failed to form tubular vacuoles, and the mutant of a putative vacuolar fusion factor, VESICLE TRANSPORT THROUGH INTERACTION WITH T-SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN ATTACHMENT PROTEIN RECEPTORS (SNARES) 11 (VTI11), could not flexibly rearrange the vacuolar network. Both mutants failed to exchange the apical and basal vacuolar contents and to polarly migrate the vacuoles, resulting in a more symmetric division of zygotes. Additionally, we observed that in contrast to sgr2, the zygotic defects of vti11 were rescued by the pharmacological depletion of phosphatidylinositol 3-phosphate (PI3P), a distinct phospholipid in the vacuolar membrane. Thus, SGR2 and VTI11 have individual sites of action in zygotic vacuolar membrane processes. Further, a mutant of YODA (YDA) mitogen-activated protein kinase kinase kinase, a core component of the embryonic axis formation pathway, generated the proper vacuolar network; however, it failed to migrate the vacuoles toward the basal region, which suggests impaired directional cues. Overall, we conclude that SGR2- and VTI11-dependent vacuolar exchange and YDA-mediated directional migration are necessary to achieve polar vacuolar distribution in the zygote.

Intracellular dynamics and transcriptional regulations in plant zygotes: a case study of Arabidopsis.

KEY MESSAGE: Recent understandings ofArabidopsiszygote. Body axis formation is essential for the proper development of multicellular organisms. The apical-basal axis in Arabidopsis thaliana is determined by the asymmetric division of the zygote, following its cellular polarization. However, the regulatory mechanism of zygote polarization is unclear due to technical issues. The zygote is located deep in the seed (ovule) in flowers, which prevents the living dynamics of zygotes from being observed. In addition, elucidation of molecular pathways by conventional forward genetic screens was not enough because of high gene redundancy in early development. Here, we present a review introducing two new methods, which have been developed to overcome these problems. Method 1: the two-photon live-cell imaging method provides a new system to visualize the dynamics of intracellular structures in Arabidopsis zygotes, such as cytoskeletons and vacuoles. Microtubules form transverse rings and control zygote elongation, while vacuoles dynamically change their shapes along longitudinal actin filaments and support polar nuclear migration. Method 2: the transcriptome method uses isolated Arabidopsis zygotes and egg cells to reveal the gene expression profiles before and after fertilization. This approach revealed that de novo transcription occurs extensively and immediately after fertilization. Moreover, inhibition of the de novo transcription was shown to sufficiently block the zygotic division, thus indicating a strong possibility that yet unidentified zygote regulators can be found using this transcriptome approach. These new strategies in Arabidopsis will help to further our understanding of the fundamental principles regarding the proper formation of plant bodies from unicellular zygotes.

Live-Cell Imaging of Zygotic Intracellular Structures and Early Embryo Pattern Formation in Arabidopsis thaliana.

Plant embryogenesis begins with fertilization and ends with the generation of the basic body plan of the future plant. Despite its importance, the dynamics of flowering plant ontogeny have long been a mystery, because the embryo develops deep in the maternal tissue. Recently, an embryonic live-cell imaging system was established in Arabidopsis thaliana by developing an in vitro ovule cultivation method and utilizing two-photon excitation microscopy (2PEM), which is suitable for deep imaging. This system enabled us to visualize intracellular dynamics during zygote polarization and monitor the cell division pattern during embryogenesis from the zygote until organ formation. In this chapter, we describe a method that allows for high-resolution imaging of cytoskeletal rearrangements in the zygote and long-term tracing of embryo patterning.

Mitochondrial dynamics and segregation during the asymmetric division of Arabidopsis zygotes.

The zygote is the first cell of a multicellular organism. In most angiosperms, the zygote divides asymmetrically to produce an embryo-precursor apical cell and a supporting basal cell. Zygotic division should properly segregate symbiotic organelles, because they cannot be synthesized de novo. In this study, we revealed the real-time dynamics of the principle source of ATP biogenesis, mitochondria, in Arabidopsis thaliana zygotes using live-cell observations and image quantifications. In the zygote, the mitochondria formed the extended structure associated with the longitudinal array of actin filaments (F-actins) and were polarly distributed along the apical-basal axis. The mitochondria were then temporally fragmented during zygotic division, and the resulting apical cells inherited mitochondria at higher concentration compared to the basal cells. Further observation of postembryonic organs showed that these mitochondrial behaviours are characteristic of the zygote. Overall, our results showed that the zygote has spatiotemporal regulation that unequally distributes the mitochondria.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes.

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote.

The asymmetric cell division of the zygote is the initial and crucial developmental step in most multicellular organisms. In flowering plants, whether zygote polarity is inherited from the preexisting organization in the egg cell or reestablished after fertilization has remained elusive. How dynamically the intracellular organization is generated during zygote polarization is also unknown. Here, we used a live-cell imaging system with Arabidopsis zygotes to visualize the dynamics of the major elements of the cytoskeleton, microtubules (MTs), and actin filaments (F-actins), during the entire process of zygote polarization. By combining image analysis and pharmacological experiments using specific inhibitors of the cytoskeleton, we found features related to zygote polarization. The preexisting alignment of MTs and F-actin in the egg cell is lost on fertilization. Then, MTs organize into a transverse ring defining the zygote subapical region and driving cell outgrowth in the apical direction. F-actin forms an apical cap and longitudinal arrays and is required to position the nucleus to the apical region of the zygote, setting the plane of the first asymmetrical division. Our findings show that, in flowering plants, the preexisting cytoskeletal patterns in the egg cell are lost on fertilization and that the zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration.

Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana.

Cell proliferation is crucial to the growth of multicellular organisms, and thus the proper control of cell division is important to prevent developmental arrest or overgrowth. Nevertheless, tools for controlling cell proliferation are still poor in plant. To develop novel tools, we focused on a specific compound family, triarylmethanes, whose members show various antiproliferative activities in animals. By combining organic chemistry to create novel and diverse compounds containing the triarylmethyl moiety and biological screens based on live-cell imaging of a fluorescently labeled tobacco Bright Yellow-2 (BY-2) culture cell line (Nicotiana tabacum), we isolated (3-furyl)diphenylmethane as a strong but partially reversible inhibitor of plant cell division. We also found that this agent had efficient antiproliferative activity in developing organs of Arabidopsis thaliana without causing secondary defects in cell morphology, and induced rapid cell division arrest independent of the cell cycle stage. Given that (3-furyl)diphenylmethane did not affect the growth of a human cell line (HeLa) and a budding yeast (Saccharomyces cerevisiae), it should act specifically on plants. Taking our results together, we propose that the combination of desired chemical synthesis and detailed biological analysis is an effective tool to create novel drugs, and that (3-furyl)diphenylmethane is a specific antiproliferative agent for plants.

Microstructure characterization of defects in cubic silicon carbide using transmission electron microscopy.

Microstructures of 3C-SiC grown by chemical vapor deposition (CVD) technique on undulant silicon substrate and a further developed technique called switch-back epitaxy (SBE) were studied using transmission electron microscopy (TEM). In case of the CVD sample, the density of the stacking faults was found to be significantly decreasing along growth direction. Sites of collision of stacking faults were observed using high-resolution transmission electron microscopy. Some of the stacking faults were observed to have disappeared after colliding into each other. The stacking faults were identified to be on the same type of plane and had the same type of displacement vector not only in CVD and SBE but also in the epitaxial layer on the SBE SiC samples.