The flower buds of Arabidopsis were double-fixed, embedded in resin, sectioned into 80 nm thick and observed using transmission electron microscopy (TEM; JEM-1400). Automatic tiling was performed using the software MATLAB and the JEM Toolbox for remote control of the TEM. Automatic image merging was performed by extending the public domain software ImageJ. This wide-range high-resolution TEM image was taken at a magnification of 12,000x and merged using 5,600 images. It shows the stage at which generative cells arose after first pollen mitosis.

The complex structure of multicellular organisms is formed by arranging multiple types of cells in a specific threedimensional organization. In the development of multicellular bodies, polarity establishments are indispensable on various scales, ranging from molecular localization on the cellular scale to the body axis on the scale of individual organisms. In this special issue on polarity in developments, we compared the establishment and reconstruction of polarity in various clades, both plants and animals, aiming to discuss whether there are common properties beyond clades and/or features unique to plants. We expect that discussions in this issue will provide new aspects of the evolution of multicellular organisms, including the relation between regularity of polarity and the evolution of body plans.

Directional transport of the phytohormone auxin causes differential auxin distribution within tissues. This gradient is essential for various aspects of plant morphogenesis, such as embryogenesis, tissue patterning, and tropism. The auxin efflux carrier PIN FORMED (PIN) is responsible for such transport by being localized to a specific side of the plasma membrane. PIN exists in organisms throughout the Streptophyta, and polar auxin transport is detected in the Charophyceae algae Chara braunii. This suggests that the importance of auxin transport is conserved among land plants and charophytes. However, the morphology of land plants and charophytes is highly diversified, from complex angiosperms to simple algae without any differentiated cells. Therefore, the role of PIN and auxin transport may be different between these plants. Here, we review the current findings on the function of PIN and auxin transport reported in Charophyte, Bryophyte, and Fern. Based on these reports, we introduce three different types of auxin transport, “cell-to-environment auxin transport”, “non-canalized intercellular auxin transport”, and “canalized auxin transport”. Each of these is assumed to contribute to plant development differently. Finally, we discuss how such types of auxin transport are involved in plant morphogenesis and their relation to plant evolution.

Gametophore shoot formation from protonemal tissues in Physcomitrium patens is used as a model to investigate the evolution of shoot architecture. The establishment of cellular polarity facilitates this morphological innovation by regulating oriented cell division. Despite the discovery of molecular mechanisms relevant to the cellular polarization, it remains largely unclear how the cellular physiological conditions are adjusted to accommodate the developmental and growth regimes. Arginine metabolism has been identified as a metabolic signature that is altered during gametophore shoot formation. The functional characterization of arginine metabolism should be investigated to determine whether and how arginine metabolism contributes to gametophore shoot formation. Here, I summarize the current knowledge on the molecular mechanism regulating gametophore shoot formation in P. patens. I describe also how arginine metabolism is modified during gametophore shoot formation. Based on this information, I finally discuss the future directions to explore the role of arginine metabolism in gametophore shoot formation by delineating the dynamics of the metabolic network using an isotope labeling experiment.

Plants regulate the direction of their growth in responding to the state and changes in the environmental factors, such as light, water, temperature, and nutrients. It is well known that gravity is the signal that allows roots to grow toward underground and stems and leaves to develop above the ground. When the direction of gravity is changed by turning the plant on its side, the plant senses the change of the direction of gravity and quickly corrects its growth, with the roots moving downward and the shoots moving upward. This is called gravitropism, and is widely known as a physiological event of plants regulated by gravity signaling. Plants do not have sensory organs like the “eyes and ears” found in animals, so how do they recognize the direction of gravity? This year, it was shown that Arabidopsis LAZY1-Like (LZY) plays a crucial role in the process of recognizing amyloplast sedimentation and transmitting information on the direction of gravity. In this paper, we first explain the historical background of gravity sensing in plants, and then describe the mechanism of gravity direction recognition exhibited by Arabidopsis LZY.

The first division of the fertilized egg is the earliest asymmetric division in many angiosperms and is considered an important process that gives rise to morphologically and functionally differentiated apical and basal cells. The process of zygote development has been analyzed in detail using Arabidopsis thaliana, a model dicotyledonous plant, and the relationship between cell asymmetric division and somatic axis formation has been demonstrated. In contrast, information on fertilized egg polarity in other angiosperms is limited, and the molecular mechanisms involved in body axis formation are largely unknown. Since fertilization and embryogenesis of angiosperms occur in the embryo sac, it has been challenging to observe the developmental process of fertilized eggs. In this review, we review the knowledge about rice axis formation and introduce our recent approaches to the developmental process of fertilized eggs using in vivo and in vitro imaging.

Shaping body axes is a critical developmental process in embryos of multicellular animals. During this process, cellcell interactions and cell rearrangements occur in a coordinated manner, which results in global remodeling of the multicellular architecture. In arthropod embryos, the early developmental process leading to body axis segmentation varies at the cellular and molecular level depending on the species. Developmental studies in insect and spider model species have provided specific examples of diverse mechanisms that regulate axis formation and segmentation in arthropod embryos. However, it is difficult to address the problem of how such developmental variations arose during the course of arthropod evolution, because of technical limitations in testing evolutionary processes. In this study, we attempted to construct a mathematical model with which evolution of developmental processes realizing major features of arthropod body plans could be tested in silico. This multicellular mathematical model was constructed with some mechanical forces of cells derived from the cytoskeleton and cell contraction in addition to the forces derived from intercellular adhesion, which changes in conjunction with each cell in the direction of planar cell polarity. This study is a theoretical research based on spider embryo model to understand the evolution of developmental processes for arthropod body plans.

Symmetry in animals and plants is a fundamental aspect of the body morphology. No matter if it is animal or plant, symmetry types in individuals can be evaluated based on organ properties such as types, numbers, and arrangements. There are mainly two types of symmetry, defined by the number of symmetry axes that divide the body into two same parts. Bilateral symmetry is characterized by a single symmetry axis and radial symmetry is characterized by multiple symmetry axes. How are symmetry types diversified? To explore these mechanisms, we mainly focused on intraspecific phenotypic variations. This review broadens its scope to include animals and plants, exploring the mechanisms of symmetry diversification. By comparing animals and plants revealed both commonalities and differences in symmetry diversity mechanisms. Transition between radial symmetry and bilateral symmetry suggests need for elements that establish a new axis, often involving asymmetric positional information along the adaxial-abaxial (directive) axis, as seen in the expression of CYCLOIDEA (CYC) homologues in angiosperm flowers. In the case of sea anemones, intraspecific polymorphism suggested the siphonoglyph establishment as a potential source of positional information, similar to the role of CYC in plants. In hydrozoans with radial symmetry, the number of symmetry axes generally increases as body size expands. Nevertheless, when we focus on larger clades such as cnidarians or eudicots, the number of symmetry axes between species is maintained, suggesting additional mechanism(s) that ensure the robustness of specific numbers. Understanding these mechanisms could provide valuable insights into the evolution and robustness of symmetry in multicellular organisms.

Electron microscopes have a significantly narrower field of view than optical microscopes. To overcome this limitation, an automatic tiling and image merging method has been developed, which enables to obtain wide-range high-resolution images. Wide-range imaging of sections can be performed with either transmission electron microscopy (TEM) or scanning electron microscopy (SEM). The wide-range TEM is recommended if high magnification is desired, while the wide-range SEM is recommended if simplicity and three-dimensional construction are desired. Nowadays, it is possible to automatically acquire wide-range, three-dimensional images, and big data images are readily available. In this paper, I discuss the benefits of wide-range imaging by electron microscopy and its applications, showing actual wide-range images.

Many small endoplasmic reticulum (ER) bodies are constantly present in the lateral root cap region of Arabidopsis thaliana. We analyzed the morphology and function of these structures by a combined method of high-pressure freezing-freeze substitution (HPF-FS) and correlative light and electron microscopy (CLEM). Analysis of transformed root tips labeled with red fluorescent protein for PYK10, the major enzyme β-glucosidase of ER bodies, revealed that PYK10 is present in small ER bodies in the lateral root cap, and that PYK10 is frequently in contact with ER bodies and vacuoles from the second layer of the lateral root cap to the outer layers, and is influxed into and localized within vacuoles. Since PYK10 is not localized in the Golgi apparatus, it was transported directly from the ER to the vacuole by the ER body of the lateral root cap, without passing through the Golgi apparatus. To clarify this process, it was important to improve HPF-FS method to optimize for CLEM that can retain ultrastructure and fluorescence and can image a wide area. This paper describes the details of the combined method of HPF-FS and CLEM, its combination with immunogold staining and serial section techniques, and the new findings obtained from the method.

Basic helix-loop-helix (bHLH) transcription factors are widely conserved across eukaryotes and play a central role in development, metabolism, and other life processes in plants and animals. In land plants, bHLH transcription factors are diversified and may contribute to complex tissue formation and environmental adaptation by regulating downstream gene expression network. Molecular genetic and evolutionary developmental studies using model plants reveal the function and evolution of bHLH transcription factors that regulate tissue formation in land plants. The formation of stomata, gas-exchange structures in plant epidermis, is regulated by heterodimers of bHLH transcription factors belonging to subfamily Ia (e.g., SPEECHLESS, MUTE, FAMA) and subfamily IIIb (e.g., ICE1/SCREAM), and this regulatory mechanism is conserved in land plants. We found that Ia and IIIb bHLHs are still retained in the stomata-less bryophyte Marchantia polymorpha, and that these heterodimers regulate the formation of a unique diploid tissue known as the seta. In this article, we discuss the functional differentiation and co-option of bHLH transcription factors in plant tissue formation and the evolution of gene expression regulation by bHLH homodimers and heterodimers, using the Ia-IIIb bHLH transcription factor module as an example.

The stem of vascular plants serves as foundational support for aboveground growth. Tissue response to mechanical forces is crucial for directing plant morphogenesis and, thus, stem growth and shape. This process plays a pivotal role in expanding the size of aboveground organs while also dictating the spatial arrangement of leaves and flowers. However, external forces can sometimes result in tissue or organ breakage. Hence, the stem must integrate forces beyond a certain threshold to control its development. In this minireview, I will focus on recent findings on Arabidopsis thaliana lines displaying deep cracks in their inflorescence stems and their research backbones are summarized. These recent studies reveal a more complex picture than previously considered regarding the relative contributions of vascular and epidermal tissues to stem mechanics and growth.

Spatiotemporal regulation of transcription plays important roles in the dynamic regulation of various biological activities. However, rapid transcriptional changes are difficult to observe in living cells. Our group established a new system for imaging transcription activity, focusing on the RNA polymerase II (RNAPII). RNAPII is a large multiprotein complex that transcribes DNA into precursors of messenger RNA. The C-terminal domain of RNAPII contains highly conserved heptad repeats that undergo several post-translational modifications during the transcription cycle. Phosphorylation of the second Ser residue (Ser2P) in the repeat signals transcription elongation and termination, making Ser2P an important indicator of the transcription. To monitor Ser2P levels in living cells, a genetically encoded system termed modification-specific intracellular antibody (mintbody) is introduced into Arabidopsis thaliana. Immunostaining and ChIP-seq analysis confirm the function of the mintbody as an intracellular antibody for Ser2P (Ser2P-mintbody). Ser2P-mintbody can change its localization in response to rapid changes in transcriptional activities under the conditions of transcription inhibitor treatment. For quantitative measurement of endogenous Ser2P levels, a two-component system is being developed. This system allows quantitative tracking of transcriptional dynamics, such as the dynamic change of transcription level during the mitotic phase. In addition, Ser2P-mintbody is useful for exploring tissue- or cell-type-specific variation of transcription activity. The observation of Ser2P-mintbody in pollen leads to the discovery of the interesting distribution pattern of the transcription active region in sperm nuclei. The approach is effective for achieving live visualization of the transcription level and facilitates a better understanding of cellular phenomena and tissue development.

Pollen tubes are essential for the reproductive processes of angiosperms and have been the subject of much research. The molecules responsible for pollen tube germination, growth, and directional control were identified. Recently, we proposed a new hypothesis of when the genes encoding these proteins are expressed and how pollen-tube capabilities are acquired. In this review, we discuss the results of recent studies in this field with a particular focus on the timing of gene expression in Arabidopsis pollen tubes. Based on this review, future prospects for pollen tube research in plants are also discussed.