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![Live-cell imaging and quantification of zygotic mitochondria. (a and b) Two-photon excitation microscopy (2PEM) images of the egg cell (a) and the time-lapse observation of the zygote in the in vitro-cultivated ovules (b) expressing the mitochondrial/nuclear marker. Maximum intensity projection (MIP) images are shown. Images are representative of nine time-lapse observations. Numbers indicate the time (h:min) from when the zygote started elongation. Yellow arrowheads indicate the nuclei, and the inset shows an enlarged image of the basal cell region. (c) Illustrations showing a summary of the respective stages. (d-f) Image processing for quantification analysis. (d) MIP image generated by serial optical sections of 2PEM images of a mature zygote expressing the mitochondrial/nuclear marker. (e) Mask image of the cell area. (f) Binary image of the mitochondria. (g-i) Graphs of the cell area (g), the total mitochondrial area (h) and the mitochondrial occupancy (i) in the young and mature zygotes. Right illustrations show the correlation between the values and the features of cells and/or mitochondria. Significant difference was determined by Mann-Whitney U test; * p < .05; * * p < .01 [n = 8 (young) and 10 (mature)]. Scale bars: 10 and 1 µm (insets).](profile/Yusuke-Kimata-2/publication/346511785/figure/fig1/AS:963933443719171@1606831050493/Live-cell-imaging-and-quantification-of-zygotic-mitochondria-a-and-b-Two-photon.jpg)
Live-cell imaging and quantification of zygotic mitochondria. (a and b) Two-photon excitation microscopy (2PEM) images of the egg cell (a) and the time-lapse observation of the zygote in the in vitro-cultivated ovules (b) expressing the mitochondrial/nuclear marker. Maximum intensity projection (MIP) images are shown. Images are representative of nine time-lapse observations. Numbers indicate the time (h:min) from when the zygote started elongation. Yellow arrowheads indicate the nuclei, and the inset shows an enlarged image of the basal cell region. (c) Illustrations showing a summary of the respective stages. (d-f) Image processing for quantification analysis. (d) MIP image generated by serial optical sections of 2PEM images of a mature zygote expressing the mitochondrial/nuclear marker. (e) Mask image of the cell area. (f) Binary image of the mitochondria. (g-i) Graphs of the cell area (g), the total mitochondrial area (h) and the mitochondrial occupancy (i) in the young and mature zygotes. Right illustrations show the correlation between the values and the features of cells and/or mitochondria. Significant difference was determined by Mann-Whitney U test; * p < .05; * * p < .01 [n = 8 (young) and 10 (mature)]. Scale bars: 10 and 1 µm (insets).
Source publication
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...
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... the filtered images were binarized by the Otsu's algorithm. e cell areas were manually determined based on the cell outline (Figure 1e), and the binary images were masked with the cell area (Figure 1f). Mitochondrial occupancy was defined and calculated as the ratio of the total mitochondrial area to the cell area. ...
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... the filtered images were binarized by the Otsu's algorithm. e cell areas were manually determined based on the cell outline (Figure 1e), and the binary images were masked with the cell area (Figure 1f). Mitochondrial occupancy was defined and calculated as the ratio of the total mitochondrial area to the cell area. ...
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... perform live-cell imaging of the mitochondria in Arabidopsis zygotes, we combined the green-fluorescent reporter visualizing mitochondria (DD45p::mt-GFP) and the red-fluorescent reporter labelling nucleus (EC1p::H2B-tdTomato and DD22p::H2B-mCherry) ( Kimata et al., 2019;Yamaoka et al., 2011). is dualcolour mitochondrial/nuclear marker was imaged using 2PEM (Figure 1a, b, and Supplementary Movie S1). ...
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... fertilization, the mitochondria were in a mesh-like pattern outside of the cellular centre (Figure 1a, c), probably because this region was occupied by large vacuoles ( Kimata et al., 2019;. Aer fertilization, the mesh-like pattern was lost, and the mitochondria were detected in the cell centre (Figure 1b, c), as the large vacuoles had shrunk (Faure et al., 1992;Kimata et al., 2019). ...
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... fertilization, the mitochondria were in a mesh-like pattern outside of the cellular centre (Figure 1a, c), probably because this region was occupied by large vacuoles ( Kimata et al., 2019;. Aer fertilization, the mesh-like pattern was lost, and the mitochondria were detected in the cell centre (Figure 1b, c), as the large vacuoles had shrunk (Faure et al., 1992;Kimata et al., 2019). en the cell elongated along the apical-basal axis, and the total mitochondrial area seemed to gradually increase (Figure 1b,c, and Supplementary Movie S1). ...
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... fertilization, the mesh-like pattern was lost, and the mitochondria were detected in the cell centre (Figure 1b, c), as the large vacuoles had shrunk (Faure et al., 1992;Kimata et al., 2019). en the cell elongated along the apical-basal axis, and the total mitochondrial area seemed to gradually increase (Figure 1b,c, and Supplementary Movie S1). ...
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... then began quantifying the changes in zygotic and mitochondrial areas. To distinguish between individual mitochondria, we acquired images with a higher spatial resolution than that used for Figure 1a, b (seven times the xy resolution and four times the z resolution), and used the images (Figure 1d) to extract the cell area (Figure 1e) and the mitochondria (Figure 1f). e cell and total mitochondrial areas were both higher in mature zygotes than in young zygotes (Figure 1g, h). e mitochondrial occupancy was slightly decreased in mature zygotes (Figure 1i), which is consistent with our previous finding that the zygote quickly expands with massive vacuole swelling ( Kimata et al., 2019). ...
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... then began quantifying the changes in zygotic and mitochondrial areas. To distinguish between individual mitochondria, we acquired images with a higher spatial resolution than that used for Figure 1a, b (seven times the xy resolution and four times the z resolution), and used the images (Figure 1d) to extract the cell area (Figure 1e) and the mitochondria (Figure 1f). e cell and total mitochondrial areas were both higher in mature zygotes than in young zygotes (Figure 1g, h). e mitochondrial occupancy was slightly decreased in mature zygotes (Figure 1i), which is consistent with our previous finding that the zygote quickly expands with massive vacuole swelling ( Kimata et al., 2019). ...
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... then began quantifying the changes in zygotic and mitochondrial areas. To distinguish between individual mitochondria, we acquired images with a higher spatial resolution than that used for Figure 1a, b (seven times the xy resolution and four times the z resolution), and used the images (Figure 1d) to extract the cell area (Figure 1e) and the mitochondria (Figure 1f). e cell and total mitochondrial areas were both higher in mature zygotes than in young zygotes (Figure 1g, h). e mitochondrial occupancy was slightly decreased in mature zygotes (Figure 1i), which is consistent with our previous finding that the zygote quickly expands with massive vacuole swelling ( Kimata et al., 2019). ...
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... then began quantifying the changes in zygotic and mitochondrial areas. To distinguish between individual mitochondria, we acquired images with a higher spatial resolution than that used for Figure 1a, b (seven times the xy resolution and four times the z resolution), and used the images (Figure 1d) to extract the cell area (Figure 1e) and the mitochondria (Figure 1f). e cell and total mitochondrial areas were both higher in mature zygotes than in young zygotes (Figure 1g, h). e mitochondrial occupancy was slightly decreased in mature zygotes (Figure 1i), which is consistent with our previous finding that the zygote quickly expands with massive vacuole swelling ( Kimata et al., 2019). ...
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... distinguish between individual mitochondria, we acquired images with a higher spatial resolution than that used for Figure 1a, b (seven times the xy resolution and four times the z resolution), and used the images (Figure 1d) to extract the cell area (Figure 1e) and the mitochondria (Figure 1f). e cell and total mitochondrial areas were both higher in mature zygotes than in young zygotes (Figure 1g, h). e mitochondrial occupancy was slightly decreased in mature zygotes (Figure 1i), which is consistent with our previous finding that the zygote quickly expands with massive vacuole swelling ( Kimata et al., 2019). ...
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... distinguish between individual mitochondria, we acquired images with a higher spatial resolution than that used for Figure 1a, b (seven times the xy resolution and four times the z resolution), and used the images (Figure 1d) to extract the cell area (Figure 1e) and the mitochondria (Figure 1f). e cell and total mitochondrial areas were both higher in mature zygotes than in young zygotes (Figure 1g, h). e mitochondrial occupancy was slightly decreased in mature zygotes (Figure 1i), which is consistent with our previous finding that the zygote quickly expands with massive vacuole swelling ( Kimata et al., 2019). ...
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... the live-cell observation, we noticed that mitochondria in elongating zygotes formed long filamentous structures along the apical-basal axis (Figure 1b, c, and Supplementary Movie S1). We therefore measured the average angle of the mitochondria against the cell longitudinal axis (∆θ) (Figure 2a-d). ...
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... therefore measured the average angle of the mitochondria against the cell longitudinal axis (∆θ) (Figure 2a-d). e basal bottom of the zygote was oen bent (Figure 1d), so we excluded this site and focussed on the cell's centre region (Figure 2c, grey circle). e mitochondrial angle in the mature zygote was significantly smaller than in the young zygote, showing the longitudinal direction of mitochondrial filaments (Figure 2d). ...
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... the time-lapse movies of the zygote, we also noticed that many dotted mitochondria were observed instead of the longitudinal filaments during cell division (Figure 1b, c, and Supplementary Movie S1); the filamentous structures were detected again aer zygotic division was completed. To further focus on this temporal mitochondrial fragmentation, we increased the time resolution. ...
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... the time-lapse images, the time frames before, during, and aaer the zygotic division were used as mature, dividing and one-cell embryo, respectively. (d) Graph of the total mitochondrial area, measured using the whole-cell areas like in Figure 1h. The le illustration of b shows a schematic representation of the Feret's diameter, and the right illustrations show the correlation between the mitochondrial features and respective values. ...
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Citations
... The inner cell mass then specifies the vascular and ground tissues, while the outer layer differentiates into the protoderm (epidermis precursor) during subsequent embryogenesis (3). Live-cell imaging has revealed that dynamic cellular changes are the driving force of zygote polarization along the apical-basal axis (4)(5)(6)(7). For example, the zygote elongates longitudinally in a manner of tip growth, and the nucleus migrates to the apical cell tip to establish the asymmetric cell division site (8,9). ...
Plants develop along apical-basal and radial axes. In Arabidopsis thaliana, the radial axis becomes evident when the cells of the eight-cell proembryo divide periclinally, forming inner and outer cell layers. Although changes in cell polarity or morphology likely precede this oriented cell division, the initial events and the factors regulating radial axis formation remain elusive. Here, we report that three transcription factors belonging to class IV homeodomain-leucine zipper (HD-ZIP IV) family redundantly regulate radial pattern formation: HOMEODOMAIN GLABROUS11 (HDG11), HDG12, and PROTODERMAL FACTOR2 (PDF2). The hdg11 hdg12 pdf2 triple mutant failed to undergo periclinal division at the eight-cell stage and cell differentiation along the radial axis. Live-cell imaging revealed that this failure in radial axis formation can be traced back to the behavior of the embryo initial cell (apical cell), which is generated by zygote division. In the wild type, the apical cell grows longitudinally and then radially and its nucleus remains at the bottom of the cell, where the vertical cell plate emerges. By contrast, the mutant apical cell elongates longitudinally and its nucleus releases from its basal position, resulting in a transverse division. Computer simulations based on the live-cell imaging data confirmed the importance of the geometric rule (the minimal plane principle and nucleus-passing principle) in determining the cell division plane. We propose that HDG11, HDG12, and PDF2 promote apical cell polarization, i.e., radial cell growth and basal nuclear retention, as the initial event of radial axis formation during embryogenesis.
... From the onset of cell elongation to just before asymmetric cell division, Arabidopsis zygotes undergo cell elongation with nuclear migration 16 (Fig. 1a). To evaluate the distribution of intracellular structures along the apical-basal axis during this period, we first acquired the time-lapse 3D images of actin filaments, mitochondria, microtubules, and vacuolar membranes in Arabidopsis zygotes from temporal cell disorganization just after fertilization to after asymmetric cell division using two-photon excitation microscopy 8,9,11 (Fig. 1b). Then, time frames from the onset of cell elongation and just before asymmetric cell division were manually determined and trimmed. ...
... The clustering analysis based on the obtained distribution information revealed that Arabidopsis zygotes may be compartmentalized from the onset of cell elongation to just before asymmetric cell division, featuring an enrichment of microtubules in apical regions and actin filaments and vacuoles in basal regions (Figs. 3 and 4). Our findings are consistent with previous observations of individual probes, such as a cortical microtubule band at the apical region 8 , large vacuoles, and actin filaments that regulate vacuolar shape at the basal region 9,10 , and a dispersed distribution of mitochondria 11 . We constructed a comprehensive view of zygote polarization orchestrated by these intracellular structures. ...
... The actin filament marker was EGG CELL1 (EC1)p::Lifeact-Venus 35 , and the microtubule marker was EC1p::Clover-TUBULIN ALPHA6 (TUA6) 8 . The mitochondrial marker (DD45p::mt-GFP) 36 was previously described 11 . The vacuolar membrane marker (EC1p::VACUOLAR H + -PPASE (VHP1)-mGFP) was previously described 9 . ...
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.
... Mitochondrial fusion unites mitochondrial genes and gene products needed for biogenesis of functional mitochondria [53,54]. This is observed in de-differentiating mesophyll protoplasts [55], shoot apical meristems [56], zygotes [57] and germinating embryos [58]. The mitochondrial fusion observed in maize YP potentially supports the accumulation of respiratory proteins observed during pollen maturation (Fig. 7). ...
Background
Cytoplasmic male sterility (CMS) is a maternally inherited failure to produce functional pollen that most commonly results from expression of novel, chimeric mitochondrial genes. In Zea mays, cytoplasmic male sterility type S (CMS-S) is characterized by the collapse of immature, bi-cellular pollen. Molecular and cellular features of developing CMS-S and normal (N) cytoplasm pollen were compared to determine the role of mitochondria in these differing developmental fates.
Results
Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed both chromatin and nuclear fragmentation in the collapsed CMS-S pollen, demonstrating a programmed cell death (PCD) event sharing morphological features with mitochondria-signaled apoptosis in animals. Maize plants expressing mitochondria-targeted green fluorescent protein (GFP) demonstrated dynamic changes in mitochondrial morphology and association with actin filaments through the course of N-cytoplasm pollen development, whereas mitochondrial targeting of GFP was lost and actin filaments were disorganized in developing CMS-S pollen. Immunoblotting revealed significant developmental regulation of mitochondrial biogenesis in both CMS-S and N mito-types. Nuclear and mitochondrial genome encoded components of the cytochrome respiratory pathway and ATP synthase were of low abundance at the microspore stage, but microspores accumulated abundant nuclear-encoded alternative oxidase (AOX). Cytochrome pathway and ATP synthase components accumulated whereas AOX levels declined during the maturation of N bi-cellular pollen. Increased abundance of cytochrome pathway components and declining AOX also characterized collapsed CMS-S pollen. The accumulation and robust RNA editing of mitochondrial transcripts implicated translational or post-translational control for the developmentally regulated accumulation of mitochondria-encoded proteins in both mito-types.
Conclusions
CMS-S pollen collapse is a PCD event coincident with developmentally programmed mitochondrial events including the accumulation of mitochondrial respiratory proteins and declining protection against mitochondrial generation of reactive oxygen species.
... In addition, a reliable ovule cultivation system for time-lapse observations has been established to record the developmental time course of growing zygotes (12)(13)(14). Using high-resolution microscopy, the ovule cultivation system can be utilized to evaluate pharmacological effects on zygotic division and monitor intracellular dynamics (15)(16)(17). In this study, we introduced an ovule cultivation system to screen compounds that affect zygotic cell division. ...
Cell division is essential for growth and development and involves events such as spindle assembly, chromosome separation, and cell plate formation. In plants, the tools used to control these events at the desired time are still poor because the genetic approach is ineffective owing to a high redundancy and lethality, as well as harmful side effects. Accordingly, we screened cell division-affecting compounds, with a focus on Arabidopsis thaliana zygotes, which individually develop in maternal ovules; the cell division was reliably traceable without time-lapse observations. We then identified the target events of the identified compounds using tobacco BY-2 cells for live-cell imaging and proteomics. As a result, we isolated two compounds, PD-180970 and PP2. PD-180970 disrupts microtubule (MT) organization and, thus, nuclear separation, presumably by inhibiting MT-associated proteins (MAP70). PP2 affected class II Kinesin-12 localization at the phragmoplast emerging site and impaired cytokinesis. Moreover, neither chemical caused irreversible damage to viability but they were effective in multiple plant species such as cucumber ( Cucumis sativus ) and moss ( Physcomitrium patens ). We propose that the combination of chemical screening based on Arabidopsis zygotes and target event specification focusing on tobacco BY-2 cells can be used to effectively identify novel tools and transiently control specific cell division events that are conserved in diverse plant species.
... The egg cell has a mix of punctate and tubular mitochondria. However, the zygote has extremely long tubular mitochondria, most likely due to MMF [83]. These long tubular mitochondria form in association with long F-actin filaments. ...
... Nucleoids are present in 90% of the promitochondria. It is feasible that the MMF that occurs in the SAM [10,82] and in the zygote [83] contributes to the nucleoid content being less heterogeneous in the promitochondrial population. During germination, there is MMF in the form of tubuloreticular mitochondria that surround the nucleus, similar to what occurs in the SAM. ...
... This means that it provides an important opportunity for all the subgenomes to interact for recombination and DNA repair for the next generation. What is known currently is that MMF occurs in the SAM [10,82] where flowering is initiated, in the zygote [83] and in germination [86], which are key points in the life cycle. This is not to say that the fusion/fission cycle involving few mitochondria is not unimportant in the cell cycle, cell development and the functioning of the cell. ...
Plant mitochondria have large genomes to house a small number of key genes. Most mitochondria do not contain a whole genome. Despite these latter characteristics, the mitochondrial genome is faithfully maternally inherited. To maintain the mitochondrial genes—so important for energy production—the fusion and fission of mitochondria are critical. Fission in plants is better understood than fusion, with the dynamin-related proteins (DRP 3A and 3B) driving the constriction of the mitochondrion. How the endoplasmic reticulum and the cytoskeleton are linked to the fission process is not yet fully understood. The fusion mechanism is less well understood, as obvious orthologues are not present. However, there is a recently described gene, MIRO2, that appears to have a significant role, as does the ER and cytoskeleton. Massive mitochondrial fusion (MMF or hyperfusion) plays a significant role in plants. MMF occurs at critical times of the life cycle, prior to flowering, in the enlarging zygote and at germination, mixing the cells’ mitochondrial population—the so-called “discontinuous whole”. MMF in particular aids genome repair, the conservation of critical genes and possibly gives an energy boost to important stages of the life cycle. MMF is also important in plant regeneration, an important component of plant biotechnology.
... Asymmetric cell divisions, o en considered formative divisions, are key to tissue formation, and require that resulting daughter cells not only differ in morphology, but most importantly possess distinct identities (Scheres & Benfey, 1999). Acquisition of different identities is preceded by establishment of cellular polarity reflected in segregation of subcellular components such as hormones, mRNA, proteins and organelles between two daughter cells, and can be influenced by both intrinsic and extrinsic mechanisms (Dong et al., 2009;Kimata et al., 2016;Kimata et al., 2019;Kimata et al., 2020). Intrinsic regulation relies on activation of cell type-specific transcriptional networks, while extrinsic control is determined by cellular environment and involves cell-to-cell communication through different mobile molecular signals such as short miRNA, short peptides, hormones, as well as mechanical cues (Heisler et al., 2010;Schlereth et al., 2010). ...
... Achievement of these goals became feasible with application of an in vitro ovule cultivation system allowing tracking of early embryogenesis events (Gooh et al., 2015). Livecell imaging was also successfully used to study mitochondria, vacuole and cytoskeleton organisation of in vitro cultivated Arabidopsis zygotes (Kimata et al., 2016;Kimata et al., 2019;Kimata et al., 2020). Although these live-imaging techniques provide a unique opportunity to visualise processes occurring in the embryo enclosed into developing seed, they are not without their own challenges. ...
Phenotypic diversity of flowering plants stems from common basic features of the plant body pattern with well-defined body axes, organs and tissue organisation. Cell division and cell specification are the two processes that underlie the formation of a body pattern. As plant cells are encased into their cellulosic walls, directional cell division through precise positioning of division plane is crucial for shaping plant morphology. Since many plant cells are pluripotent, their fate establishment is influenced by their cellular environment through cell-to-cell signaling. Recent studies show that apart from biochemical regulation, these two processes are also influenced by cell and tissue morphology and operate under mechanical control. Finding a proper model system that allows dissecting the relationship between these aspects is the key to our understanding of pattern establishment. In this review, we present the Arabidopsis embryo as a simple, yet comprehensive model of pattern formation compatible with high-throughput quantitative assays.
The complex structures of multicellular organisms originate from a unicellular zygote. In most angiosperms, including Arabidopsis thaliana, the zygote is distinctly polar and divides asymmetrically to produce an apical cell, which generates the aboveground part of the plant body, and a basal cell, which generates the root tip and extraembryonic suspensor. Thus, zygote polarity is pivotal for establishing the apical-basal axis running from the shoot apex to the root tip of the plant body. The molecular mechanisms and spatiotemporal dynamics behind zygote polarization remain elusive. However, advances in live-cell imaging of plant zygotes have recently made significant insights possible. In this Cell Science at a Glance article and the accompanying poster, we summarize our understanding of the early steps in apical-basal axis formation in Arabidopsis, with a focus on de novo transcriptional activation after fertilization and the intracellular dynamics leading to the first asymmetric division of the zygote.
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.
Seed development in flowering plants is highly complex and governed by three genetically distinct tissues: the fertilization products, the diploid embryo and triploid endosperm, as well as the seed coat that has maternal origin. There are diverse cellular dynamics such as nuclear movement in gamete cells for fertilization, cell polarity establishment for embryo development, and multinuclear endosperm formation. These tissues also coordinate and synchronize the developmental timing for proper seed formation through cell-to-cell communications. Live-cell imaging using advanced microscopy techniques enables us to decipher the dynamics of these events. Especially, the establishment of a less-invasive semi-in vivo live-cell imaging approach has allowed us to perform time-lapse analyses for long period observation of Arabidopsis thaliana intact seed development dynamics. Here we highlight the recent trends of live-cell imaging for seed development and discuss where we are heading.
