Tomokazu Kawashima

Using Genomics to Study Legume Seed Development

Seeds are essential for flowering plant reproduction because they protect, nourish, and contain the developing embryo that represents the next sporophytic generation. In addition, seeds contain energy resources that sustain the young sporophyte during germination before photosynthesis begins. In

The suspensor: not just suspending the embryo

The suspensor is a terminally differentiated embryonic region that connects the embryo to surrounding tissues during early seed development. Most seed-bearing plant embryos contain suspensor regions, which occur in a wide variety of sizes and shapes, and suspensor-like structures are present in the embryos of some lower land plants. Recent technological advances, including novel genomics approaches, have provided insights into the function of the suspensor and the DNA sequences that control suspensor-specific gene expression. The molecular mechanisms controlling embryo basal-cell lineage specification and suspensor differentiation events are also beginning to be illuminated. Here, we summarize the role of the suspensor in plant embryogenesis and discuss future directions of suspensor biology, including the dissection of suspensor gene regulatory networks.

Epigenetic reprogramming in plant sexual reproduction.

Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation.

The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis.

Histone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

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.

Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana

In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin–myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants. DOI: http://dx.doi.org/10.7554/eLife.04501.001

Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote

Significance In animals and plants, the zygote divides unequally, and the daughter cells inherit different developmental fates to form a proper embryo along the body axis. The cytological events leading to zygote polarization have remained unknown in flowering plants. Here, we report that the two essential components of the cytoskeleton, microtubules and actin filaments, are both disorganized on fertilization and then, arranged to form a transverse ring leading directional cell elongation and longitudinal arrays underlying polar nuclear migration, respectively. These results provide insights into the intracellular dynamics of zygote and the specific roles of cytoskeletons on zygote polarization in flowering plants. 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.

Identification of cis-regulatory sequences that activate transcription in the suspensor of plant embryos

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the scarlet runner bean (Phaseolus coccineus) G564 gene to understand how genes are activated specifically within the suspensor during early embryo development. Previously, we showed that the G564 upstream region has a block of tandem repeats, which contain a conserved 10-bp motif (GAAAAGC/TGAA), and that deletion of these repeats results in a loss of suspensor transcription. Here, we use gain-of-function (GOF) experiments with transgenic globular-stage tobacco embryos to show that only 1 of the 5 tandem repeats is required to drive suspensor-specific transcription. Fine-scale deletion and scanning mutagenesis experiments with 1 tandem repeat uncovered a 54-bp region that contains all of the sequences required to activate transcription in the suspensor, including the 10-bp motif (GAAAAGCGAA) and a similar 10-bp-like motif (GAAAAACGAA). Site-directed mutagenesis and GOF experiments indicated that both the 10-bp and 10-bp-like motifs are necessary, but not sufficient to activate transcription in the suspensor, and that a sequence (TTGGT) between the 10-bp and the 10-bp-like motifs is also necessary for suspensor transcription. Together, these data identify sequences that are required to activate transcription in the suspensor of a plant embryo after fertilization.

Diversification of histone H2A variants during plant evolution

Among eukaryotes, the four core histones show an extremely high conservation of their structure and form nucleosomes that compact, protect, and regulate access to genetic information. Nevertheless, in multicellular eukaryotes the two families, histone H2A and histone H3, have diversified significantly in key residues. We present a phylogenetic analysis across the green plant lineage that reveals an early diversification of the H2A family in unicellular green algae and remarkable expansions of H2A variants in flowering plants. We define motifs and domains that differentiate plant H2A proteins into distinct variant classes. In non-flowering land plants, we identify a new class of H2A variants and propose their possible role in the emergence of the H2A.W variant class in flowering plants.

Green love talks; cell–cell communication during double fertilization in flowering plants

A major breakthrough in understanding double fertilization has been made by high resolution live-imaging. This has helped resolve several disputed issues such as preferential fertilization and polyspermy block. Cumulated information of molecular components involved in double fertilization highlights the importance of cell-cell communication between male and female gametophytes.