The wild-type unpollinated Arabidopsis pistil was fixed with 4% paraformaldehyde (w/v) for 1 h and then cleared in ClearSee.v2 solution for 4 weeks. To observe the internal structure of the pistil, it was stained with 0.1% (w/v) calcofluor staining solution after clearing. This two-photon image was taken using a two-photon excitation microscope (A1R MP, Nikon) at an excitation wavelength of 850 nm. It shows the longitudinal optical section of the ovary, including each side of the ovary wall, the central transmitting tract, and the vertically aligned ovules on both sides of the transmitting tract. The width of this image is approximately 500 µm.

In plant cells, organelles actively interact with each other. Organelles such as plastids and mitochondria originally evolved through the symbiosis of different organisms, and their dynamics are symbolic of the interactions between organisms. Plants still face daily attacks from microorganisms, but at the same time, various plant-microbe interactions, such as symbiotic relationships, are taking place. Recent imaging techniques have made it possible to capture such interactions in situ. At this symposium, held at the 88^th Annual Meeting of the Botanical Society of Japan, speakers conducting research on organelle-organelle and microbe-plant interactions using the latest imaging techniques of optical and electron microscopy gave lectures. By introducing examples of their analyses, discussions were held from a new perspective of imaging "interactions."

In the male gametophyte of plants, organelle interactions that are temporally specific, dynamic, and unique have been observed. In the microspores and male gametophytes of Pharbitis nil, mitochondria completely surround the nucleus at a certain stage. In the male gametophyte of Arabidopsis thaliana, lipid bodies encircle the generative cell, and as maturation progresses, vacuoles surround the sperm cells, exhibiting dynamic changes. Additionally, a process known as microlipophagy, in which vacuoles contact lipid bodies, undergo membrane fusion, internalize, and degrade them, has been observed. In recent years, the existence of membrane contact sites (MCS), where organelles come into close proximity and exchange materials, has been increasingly recognized. This paper reinterprets organelle interactions observed in the male gametophyte of plants from the perspective of MCS and discusses the complexity and diversity of inter-organelle interactions.

Organelles in eukaryotic cells not only perform individual functions but also form regulatory systems that require coordinated interactions among multiple organelles to carry out various biological processes. For example, many metabolic systems depend on spatiotemporally regulated organelle-organelle interactions to facilitate the exchange of metabolites between organelles. Peroxisomes, which are ubiquitously present in eukaryotic cells, play key roles in processes such as fatty acid metabolism, reactive oxygen species (ROS) detoxification, photorespiration, and the biosynthesis of jasmonic acid. Defects in these functions can lead to seed germination failure, dwarfism, reproductive abnormalities, and embryonic lethality. Photorespiration, a metabolic pathway involving peroxisomes, chloroplasts, and mitochondria, requires the light-dependent interaction of these three organelles. During this process, changes in peroxisome morphology and increased adhesion to chloroplasts occur. Additionally, during the germination of oilseed plants, fatty acids produced by lipase-mediated hydrolysis of triacylglycerol in the oil body are transported to peroxisomes, where direct interaction between peroxisomes and oil bodies takes place. It is now clear that these organelle-organelle interactions occur via membrane contact sites, facilitating lipid transport between organelles. This review focuses on the molecular mechanisms underlying the interactions between peroxisomes and other organelles, with a particular emphasis on studies in Arabidopsis thaliana.

Organelles compartmentalize specific sets of enzymes and metabolites, enabling them to perform their characteristic functions. Due to this functional specialization, organelles have long been considered spatially independent structures. However, recent research has identified membrane contact sites (MCS), regions where different organelle membranes are directly connected, revealing that organelles collaborate more closely than previously thought. The discovery of MCS has introduced a paradigm shift in organelle research, offering a new perspective on how organelles function. In this article, I will provide an overview of the latest trends in MCS research, with a particular focus on the mechanisms of lipid transport via MCS in yeast.

Most land plants grow roots in the soil and interact with various soil microorganisms throughout their life. Legumes, such as soybeans, form symbiotic structures called root nodules through interaction with rhizobia. Microscopic observations have advanced our understanding of rhizobial infection processes. However, previous research has primarily relied on the destructive method of excavating roots from the soil. In this paper, we introduce the Rhizosphere Frame system, which we have developed for nondestructive observation of root nodule symbiosis in the soil. This system is expected to facilitate a deeper understanding of the dynamics of plant-microbe interactions in the soil environment.

Plant-pathogenic microbe interaction is a complex phenomenon that is initiated by the recognition of both organisms at the site of infection and dynamically changes as the plant immune system and the pathogenic microbial virulence system interact with each other. Live imaging is an important tool to correctly capture such a dynamic phenomenon that includes spatio-temporal dynamics and to understand its molecular mechanisms. Here, we present an example of our work analyzing the spatio-temporal dynamics of defense response marker gene expression.

Lichens, symbiotic organisms consisting of fungi and algae, are commonly found on various hard surfaces. Due to the challenges of preparing ultrathin sections of lichens embedded in minerals, electron microscopy (EM) has been mainly limited to surface analysis. To overcome this limitation, we present a "cross-sectional polishing SEM method". This technique involves embedding lichens in resin, cutting cross-sections with a band saw, mechanically polishing them, and observing the surface by scanning electron microscopy (SEM). By applying this method to a lichen-covered rock, we successfully obtained detailed cross-sectional images revealing the internal structure of the symbiotic algae and fungi, as well as the mineral substrate. This innovative approach provides a valuable tool for studying the internal anatomy of lichens, especially those growing on minerals.

The root cap mucilage, a gel-like substance secreted by border cells or border-like cells at the root cap, plays a crucial role in plant root adaptation to soil environments. Despite its importance, methods for analyzing root cap mucilage, especially visualization techniques, remain limited. This study introduces a simple method using fluorescent silica nanoparticles (Quartz Dot) to visualize and quantify the area of secreted mucilage with confocal laser scanning microscopy. The nanoparticles used in this study are embedded with fluorescent probes and large enough to be excluded from the apoplastic mucilage matrix, thereby outlining the boundary of the mucilage region surrounding the root cap. Combined with fluorescent probes for cell wall staining, this approach allows the quantification of the minute mucilage areas surrounding the Arabidopsis root cap. The method was validated by comparing the mucilage region in wild-type Arabidopsis and the smb brn1 brn2 mutant, revealing a significant reduction in mucilage areas in the mutant. Additionally, we investigated the effects of several abiotic stresses, including toxic ions and nutrient starvation, on the area of Arabidopsis root cap mucilage, and further identified soil stresses that promote expansion of the root cap mucilage region, underscoring the sensitivity and precision of our method. Collectively, this study established a simple and robust technique for visualizing and quantifying the root cap mucilage region. The demonstrated utility of this method indicates its potential for broader applications in understanding the physiological and molecular mechanisms underlying root cap functionalization and its role in adaptation to soil environments.

Plants maintain genetic diversity through strategies such as “dioecy,” where male flowers (♂) and female flowers (♀) are borne on separate individuals. Many dioecious plants have sex chromosomes, and in the XY system, the Y chromosome has the gene determining maleness. Sex chromosomes evolved from a pair of autosomes, with the Y chromosome diverging over time to form heteromorphic sex chromosomes, where the X and Y differ in size. Silene latifolia, a member of the Caryophyllaceae family, is a model species for studying heteromorphic sex chromosomes. Its Y chromosome contains a large recombination suppression region (~500 Mb), complicating the identification of sex-determining genes. By analyzing these chromosomes, the authors identified Gynoecium suppressing function on Y (GSFY), a gene that suppresses pistil development. GSFY was found to be an ortholog of the CLAVATA3 gene in Arabidopsis thaliana, involved in pistil reduction. Moreover, it was found that during the evolution of sex chromosomes, the X copy of the CLAVATA3 ortholog became nonfunctional, while the WUSCHEL ortholog, which is involved in pistil development, was lost from the Y chromosome but retained on the X chromosome. These findings suggest the X chromosome plays a role in sex determination, providing new insights into the evolution of plant sex chromosomes.

In the sexual reproduction of land plants, the production of female gamete egg and male gamete sperm is an essential process. These processes have drastically changed through plant evolution. In bryophytes, lycophytes, and monilophytes, multicellular gametangia develop as reproductive organs during the haploid generation. Egg and sperm are produced in the female and male gametangia, archegonia and antheridia, respectively. Until a decade ago, knowledge about gametangium development was largely limited to anatomical observations, and the molecular mechanisms underlying their development remained poorly understood. Recently, however, the establishment of research tools for the model bryophyte Marchantia polymorpha has enabled significant progress in uncovering the molecular genetics of gametangium development. In our research, we have focused on gametangium development and identified a BZR/BES transcription factor MpBZR3 as a key regulator of this process. This article will provide an overview of recent research advancements in understanding gametangium development in bryophytes.

Imaging and analysis of precise morphology and tissue structure are important for understanding the development of organisms and tissue structure. However, it is generally difficult to visualize the deep tissues of living organisms because the body of organisms is opaque. Two-photon excitation microscopy achieves deeper penetration and reduces photodamage compared to single-photon excitation, such as confocal microscopy. Therefore, it is widely used for in vivo live imaging. We have demonstrated the importance of selecting both appropriate fluorescent proteins and excitation wavelengths for clear two-photon imaging in deep plant tissues. We also developed a two-photon live imaging method, named single-locule method, to directly observe the process of pollen tube guidance and fertilization in the pistil of Arabidopsis thaliana. Spatiotemporal regulation of pollen tube growth by each cell of the female and male gametophyte and maternal tissues results in multistep polyspermy blockings that achieve successful fertilization. In addition to these recent studies, this review also provides tips on how to observe deep plant tissues in vivo and how the single-locule method was established.