in-vitro-ovule-cultivation-for-live-cell-imaging-zygote-polarization

<em>In Vitro</em> Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in <em>Arabidopsis thaliana</em>

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; ...

Nagoya University

[Maintenance of the root apical meristem].

In higher plants, post-embryonic development is dependent on the activity of the root and shoot apical meristem (RAM and SAM). The quiescent center (QC) in the RAM and the organizing center (OC) in the SAM are known to be essential for the maintenance of meristematic activity. To understand the mechanism that maintains post-embryonic meristems, we isolated an Arabidopsis mutant, halted root (hlr). In this mutant, the cellular organization was disrupted in post-embryonic meristems both in the root and in the shoot, and their meristematic activity was reduced or became abnormal. We showed that the mutant RAM lost its QC identity after germination, which was specified during embryogenesis, whereas the identity of differentiated tissues was maintained. In the post-embryonic SAM, the expression pattern of a typical OC marker gene, WUSCHEL, was disturbed in the mutant. These observations indicate that the HLR gene is essential to maintain the cellular organization and normal nature of the RAM and SAM. The HLR gene encodes RPT2a, which is a subunit of the 26S proteasome that degrades key proteins in diverse cellular processes. We showed that the HLR gene was expressed both in the RAM and in the SAM, including in the QC and the OC, respectively, and that the activity of proteasomes were reduced in the mutant. We propose that proteasome-dependent programmed proteolysis is required to maintain the meristem integrity both in the shoot and in the root.

The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems.

Recent studies using Arabidopsis propose a framework of root development and pattern formation that can be divided to three processes. First, a positional signal that is delivered from neighboring cells controls the fate of undifferentiated cells. Then, cell fate is fixed through a protein-network that includes various transcription factors. Finally, the expression of a particular gene-set leads to fate-dependent cell differentiation, resulting in oriented cell division, cell specification and cell elongation. In addition, these processes could be modified by chromatin stabilization and protein degradation. We focus on three fundamental patterns of root development, circumferential pattern, radial pattern and proximo-distal pattern, and on novel approaches to identify genes that are responsible for the spatiotemporal regulation of root development.

Stepwise understanding of root development.

Axis formation is one of the earliest patterning events in plant and animal embryogenesis. In Arabidopsis, the main axis of the embryo is evident at the asymmetric division of the zygote into a small, embryonic apical cell and a large extraembryonic basal cell. Here we show that the homeobox genes WOX2 and WOX8, which are initially coexpressed in the zygote, act as complementary cell fate regulators in the apical and basal lineage, respectively. Furthermore, WOX8 expression in the basal cell lineage is required for WOX2 expression and normal development of the proembryo, suggesting an inductive mechanism. The identified WOX cascade is required for normal expression of a reporter gene of the auxin efflux carrier PIN1 and for the formation of auxin response maxima in the proembryo. Thus, our results link the spatial separation of WOX transcription factors to localized auxin response and the formation of the main body axis in the embryo.

Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo.

In most flowering plants, the apical-basal body axis is established by an asymmetric division of the polarized zygote. In Arabidopsis, early embryo patterning is regulated by WOX homeobox genes, which are coexpressed in the zygote but become restricted to apical (WOX2) and basal (WOX8/9) cells. How the asymmetry of zygote division is regulated and connected to the daughter cell fates is largely unknown. Here, we show that expression of WOX8 is independent of the axis patterning signal auxin, but, together with the redundant gene WOX9, is activated in the zygote, its basal daughter cell, and the hypophysis by the zinc-finger transcription factor WRKY2. In wrky2 mutants, egg cells polarize normally but zygotes fail to reestablish polar organelle positioning from a transient symmetric state, resulting in equal cell division and distorted embryo development. Both defects are rescued by overexpressing WOX8, indicating that WRKY2-dependent WOX8 transcription links zygote polarization with embryo patterning.

Transcriptional activation of Arabidopsis axis patterning genes WOX8/9 links zygote polarity to embryo development.

The 26S proteasome plays fundamental roles in the degradation of short-lived regulatory proteins, thereby controlling diverse cellular processes. In Arabidopsis, the essential RPT2 subunit is encoded by two highly homologous genes: RPT2a and RPT2b. Currently, only RPT2a has been reported to regulate various developmental processes, including the maintenance of the root apical meristem (RAM), although the roles of RPT2a in the RAM are still obscure. Here, we analyzed the cell type-specific requirement for RPT2a. When RPT2a was expressed locally in the rpt2a mutant, pleiotropic defects in the RAM, such as cell death and distorted cellular organization, were rescued differently, suggesting that RPT2a regulates various specific activities, which converge to maintain the RAM. On the other hand, the homologous RPT2b was also expressed in meristems, and the expression of RPT2b protein under the control of the RPT2a promoter complemented the rpt2a RAM defects, although the rpt2b mutant showed no obvious defect in all developmental aspects we examined. These results show that RPT2b might work in the RAM, but is dispensable for RAM maintenance in the presence of RPT2a. In contrast, the rpt2a rpt2b double mutant was lethal in male and female gametophytes, suggesting that RPT2a and RPT2b are redundantly required for gametogenesis. Furthermore, we showed that similar meristematic and gametophytic defects were caused by mutations in other subunit genes, RPT5a and RPT5b, suggesting that proper activity of the proteasome, not an RPT2-specific function, is required. Taken together, our results suggest that RPT2a and RPT2b contribute differently to the proteasome activity required for each developmental context.

Arabidopsis RPT2a encoding the 26S proteasome subunit is required for various aspects of root meristem maintenance, and regulates gametogenesis redundantly with its homolog, RPT2b.

To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography.

Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.

During embryogenesis, the basic body plan of an organism develops from a unicellular zygote. In most flowering plants, the polar zygote divides asymmetrically, making visible the apical-basal axis in the early embryo. The molecular mechanisms governing how the zygote polarizes and how this polarity is linked to embryo axis formation have been obscure, mainly owing to the difficulties to access the zygote that is deeply embedded in the maternal tissue. In this review, we summarize recent findings identifying key regulators in Arabidopsis and developing novel approaches in various plant species, which altogether set the stage for unraveling embryo axis formation.

The origin of the plant body axis.

Arabidopsis SOG1 (suppressor of gamma response 1) is a plant-specific transcription factor that governs the DNA damage response. Here we report that SOG1 is phosphorylated in response to DNA damage, and that this phosphorylation is mediated by the sensor kinase ataxia telangiectasia mutated (ATM). We show that SOG1 phosphorylation is crucial for the response to DNA damage, including transcriptional induction of downstream genes, transient arrest of cell division and programmed cell death. Although the amino-acid sequences of SOG1 and the mammalian tumour suppressor p53 show no similarity, this study demonstrates that ATM-mediated phosphorylation of a transcription factor has a pivotal role in the DNA damage response in both plants and mammals.

ATM-mediated phosphorylation of SOG1 is essential for the DNA damage response in Arabidopsis.

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M-specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S-phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy-terminal region is responsible for proteasome-mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S-specific promoter of a histone 3.1-type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M-specific CYCB1-GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time-lapse imaging of cell cycle progression. The resultant dual-color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development.

A dual-color marker system for in vivo visualization of cell cycle progression in Arabidopsis.

Electron-donating aryl groups were attached to electron-accepting benzophosphole skeletons. Among several derivatives thus prepared, one benzophosphole oxide was particularly interesting, as it retained high fluorescence quantum yields even in polar and protic solvents. This phosphole-based compound exhibited a drastic color change of its fluorescence spectrum as a function of the solvent polarity, while the absorption spectra remained virtually unchanged. Capitalizing on these features, this phosphole-based compound was used to stain adipocytes, in which the polarity of subcellular compartments could then be discriminated on the basis of the color change of the fluorescence emission.

Environment-sensitive fluorescent probe: a benzophosphole oxide with an electron-donating substituent.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

Intercellular communications are essential for cell proliferation and differentiation during plant embryogenesis. However, analysis of intercellular communications in living material in real time is difficult owing to the restricted accessibility of the embryo within the flower. We established a live-embryo imaging system to visualize cell division and cell fate specification in Arabidopsis thaliana from zygote division in real time. We generated a cell-division lineage tree for early embryogenesis in Arabidopsis. Lineage analysis showed that both the direction and time course of cell division between sister cells differed along the apical-basal or radial axes. Using the Arabidopsis kpl mutant, in which single-fertilization events are frequent, we showed that endosperm development is not required for pattern formation during early embryogenesis. Optical manipulation demonstrated that damage to the embryo initial cell induces cell fate conversion of the suspensor cell to compensate for the disrupted embryo initial cell even after cell fate is specified.

Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis.

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.

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

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.

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

In many plants, the asymmetric division of the zygote sets up the apical-basal axis of the embryo. Unlike animals, plant zygotes are transcriptionally active, implying that plants have evolved specific mechanisms to control transcriptional activation of patterning genes in the zygote. In Arabidopsis, two pathways have been found to regulate zygote asymmetry: YODA (YDA) mitogen-activated protein kinase (MAPK) signaling, which is potentiated by sperm-delivered mRNA of the SHORT SUSPENSOR (SSP) membrane protein, and up-regulation of the patterning gene WOX8 by the WRKY2 transcription factor. How SSP/YDA signaling is transduced into the nucleus and how these pathways are integrated have remained elusive. Here we show that paternal SSP/YDA signaling directly phosphorylates WRKY2, which in turn leads to the up-regulation of WOX8 transcription in the zygote. We further discovered the transcription factors HOMEODOMAIN GLABROUS11/12 (HDG11/12) as maternal regulators of zygote asymmetry that also directly regulate WOX8 transcription. Our results reveal a framework of how maternal and paternal factors are integrated in the zygote to regulate embryo patterning.

Article	Year
[Maintenance of the root apical meristem]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme\| PubMed ID: 12357606	2002
The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems. Development (Cambridge, England)\| PubMed ID: 15073153	2004
Stepwise understanding of root development. Current opinion in plant biology\| PubMed ID: 15653403	2005
Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo. Developmental cell\| PubMed ID: 18539115	2008
Transcriptional activation of Arabidopsis axis patterning genes WOX8/9 links zygote polarity to embryo development. Developmental cell\| PubMed ID: 21316593	2011
Arabidopsis RPT2a encoding the 26S proteasome subunit is required for various aspects of root meristem maintenance, and regulates gametogenesis redundantly with its homolog, RPT2b. Plant & cell physiology\| PubMed ID: 21784786	2011
Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography. Journal of assisted reproduction and genetics\| PubMed ID: 22695746	2012
The origin of the plant body axis. Current opinion in plant biology\| PubMed ID: 22921364	2012
ATM-mediated phosphorylation of SOG1 is essential for the DNA damage response in Arabidopsis. EMBO reports\| PubMed ID: 23907539	2013
A dual-color marker system for in vivo visualization of cell cycle progression in Arabidopsis. The Plant journal : for cell and molecular biology\| PubMed ID: 25158977	2014
Environment-sensitive fluorescent probe: a benzophosphole oxide with an electron-donating substituent. Angewandte Chemie (International ed. in English)\| PubMed ID: 25740735	2015
Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism. Cell\| PubMed ID: 25913191	2015
Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis. Developmental cell\| PubMed ID: 26166301	2015
Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana. Plant & cell physiology\| PubMed ID: 27519314	2016
Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27911812	2016
Transcriptional integration of paternal and maternal factors in the Arabidopsis zygote. Genes & development\| PubMed ID: 28404632	2017

Minako Ueda

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