Observation and investigation of subcellular structure and of behavior of molecules are critical to analyze and to understand plant development and physiology. To achieve these observations, it is necessary not only to use but also to develop cutting-edge techniques for imaging. We have organized a symposium entitled “Frontline of imaging to understand diverse plant phenomena: from subcellular structure to environmental response” at the 80^th Annual Meeting of the Botanical Society of Japan. This symosium was co-sponsored by The Japanese Society of Plant Morphology, Integrated Imaging Research Support (IIRS), and Integrative System of Autonomous Environmental Signal Recognition and Memorization for Plant Plasticity (Scientific Research on Innovative Areas, a MEXT Grant-in Aid Project). In this symposium, six researchers have presented cutting-edge techniques for imaging such as visualizing specific molecules or structures, live-imaging method for deep inside the tissue, and quantitative evaluation of subcellular component by microscopic image analysis. Novel insights in regulating various plant phenomena obtained by using these techniques were also presented.

Histone modification regulates genome-wide gene expression. The conventional methods including chromatin immunoprecipitation and immunofluorescence microscopy require protein extraction and cell fixation. We established a live imaging method to analyze the histone modification levels in living plant cells. We used the fluorescent protein fused mintbody (modification specific intracellular antibody), which is a probe prepared based on an antibody against an acetylated amino acid of a histone. The mintbody in plant cells migrated inside and outside the nucleus and accumulated in the nucleus in proportion to the histone acetylation level.

Efficient degradation of cellulose enables us to produce fuels and chemicals from plant resources. However, biochemical conversion of cellulose by cellulase is quite slow, and the reaction becomes a bottleneck of the process. We carried out the real-time visualization of individual cellulase molecules using an advanced version of high-speed atomic force microscopy (HS-AFM), which has sub-second time resolution and nanometer space resolution. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface, but the movement of individual molecules was halted by other molecules, leading to traffic jams of enzyme molecules. This would decrease the apparent activity of the enzyme, as the hydrolytic action is coupled with molecular movement. Changing the crystalline polymorphic form of the cellulose increased the number of available lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this crystalline cellulose with another type of Trichoderma cellobiohydrolase, TrCel6A, led to further increase in the number of entrance and exit points on the substrate surface, resulting in a significant increase in the proportion of mobile TrCel7A molecules on the surface.

Visualization of cytoskeletal organizations is a fundamental research method for understanding plant cell activities. Conventionally, immunostaining methods have been used to visualize cytoskeletons, but some technical difficulties make it hard to obtain a large number of reliable microscopic images. The introduction of fluorescent protein tagging technology and the development of high-throughput method for microscopic image acquisition have made it easier and quicker to obtain many reliable microscopic digital images of cytoskeletons. Based on these technical improvements, a method for quantitative evaluation of cytoskeletal organizations by image analysis has been developed. This method has become an indispensable research approach in state-of-the-art plant cell biology. In this minireview, I outline a practical method to measure image features to quantitatively evaluate the orientation, parallelness, bundling, and density of cytoskeletons using the ImageJ image analysis software.

The basic body plan of an organism develops from a unicellular zygote. In most plants, the zygote divides asymmetrically, generating two daughter cells of different fates: the small apical daughter will form the aerial organs of the plant, whereas the large basal cell will produce the below-ground root. Therefore zygote polarization would be crucial to establish the shoot-root (apical-basal) axis of mature plant. Despite the obvious importance, how dynamically the zygote polarizes has long been obscure. In this review, I introduce the recent findings of the zygote polarization dynamics, which were obtained by using live-cell imaging of Arabidopsis thaliana. By combining image analysis and specific inhibitors, this live-cell imaging system enabled to quantify the zygotic features and to identify the driving force of zygote polarization. Here I introduce these findings to consider how we can reveal the mechanisms of plant body axis formation.

Florigen is a mobile signal that initiates flowering, which is generated in leaves in response to various environmental stimuli and is transported to the shoot apical meristem (SAM) in plants. The molecular nature of florigen was found to be proteins encoded by the gene FLOWERING LOCUS T (FT) and its orthologs. Recent progress in the molecular biology of florigen revealed its receptors and a transcriptional complex composed of florigen, receptor and transcription factors. In vivo imaging of florigen distribution in the shoot apex and inside a cell contributed to elucidate the essential mechanisms for florigen function. In rice shoot apex, distribution of florigen is clearly visualized by expression of FT protein fused with green fluorescent protein (GFP), and the spatial patterns of downstream gene expression are also visualized by various techniques. At the cellular level, the distribution of florigen and its receptor complex is observed through bimolecular fluorescent complementation (BiFC), which revealed dynamic changes of subcellular localization for florigen and related proteins during the formation of florigen-receptor complex. Here the technique for dissecting SAM is presented to show how SAM samples are prepared for imaging florigen, and recent advances in the regulation of flowering in relation to the contributions from the application of imaging techniques are summarized.

Striga hermonthica is a parasitic plant that deprives host plants of nutrients. This parasite causes the largest agricultural damage in Africa by spoiling main crops such as rice, corn, and sorghum. The agricultural losses are estimated to be 10 billion US dollars per year, making the Striga problem an issue to be urgently solved. Since the first isolation of strigolactone as a host-derived germination stimulant, the signaling mechanism of strigolactones has been the center of attention to control the Striga problem. However, the strigolactone receptor in Striga and its signaling mechanism remained elusive for a long time. Herein, we summarize recent progress in strigolactone research including the identification of the strigolactone receptors related to the Striga germination and the visualization of their function during germination. We envision that these progresses will provide a clue for elucidating the mechanism of Striga germination and, consequently, saving crop losses.

The breeding of microalgae, of which size is less than 1/10 mm, has been drawing attention. Most of the oxygen in the atmosphere, the white cliffs of Dover, and even shale gas, are all originated from microalgae propagated extensively on the earth in the geological age. This is a report for the symposium of 3aSB01-06 in the 80th Annual Meeting of the Botanical Society of Japan (16th to 19th September 2016, Okinawa) “Various attractions of microalgae: classification, evolution, genome to morphology, algal biology”. To introduce charms of microalgae, researchers with various careers, and men and women of all ages, such as Professor Kuroiwa and Dr. Mami Nomura, gave talks about systematics, evolution, genome to morphology and algal biotechnology of the microalgae.

The complete genome sequence of cyanobacteria was reported in 1990s as the first genome of photosynthetic organism. Since then, researchers have sequenced several cyanobacterial strains, and utilize them as ‘model’ organisms. However, recent emergence of the next-generation sequencers enables researchers to study diverse ‘non-model’ cyanobacteria strains. We have been studying the diversity of molecular processes of chromatic acclimation in the cyanobacteria phylum and have obtained following results: (1) an existence of the common green and red light-sensing mechanism using phytochrome-related photoreceptor, (2) an existence of two types of signal transduction pathway for chromatic acclimation, and (3) an existence of more than 4 types of gene sets of photosynthetic antenna that are regulated by light colors. Distribution of these light-sensing systems is not correlated with the phylogenetic tree produced based on the sequences of 16S ribosomal RNA, suggesting that these systems are acquired by horizontal gene transfer. Next generation sequencing technology will facilitate our understanding of diverse light-harvesting and light-sensing processes in the cyanobacteria phylum.

Energy production and cell division are one of the most important functions for living as a cell. On the other hand, micro algae and protist that are constituted with only one cell have highly diversified functions. There are many phenomena that can not be explained by our current knowledge. For example, delicate siliceous frustule formation in diatoms, rapid haptonematal coiling in haptophytes, predation using a veil in dinoflagellates, shell assembly outside the cell in testate amoebae and so on. Here, we focused on shell assembly of testate amoebae. Cell body of testate amoeba never comes out from its shell except pseudopodia. Before cell division, they form a new shell for one of two daughter cells. Interestingly, testate amoebae assemble scales into round shape using pseudopodia without casting mold outside of their cell. Paulinella chromatophora is one of the few testate amoebae that can be cultivated. We revealed the shell assembly process in P. chromatophora and discussed about the potential function of eukaryotic cell.

Pseudochoricystis ellipsoidea (MBIC11204, NBRC112353) is a unicellular green alga, which belongs to the family Trebouxiophyceae. Under nitrogen-starvation conditions, P. ellipsoidea accumulates storage lipids consisting primarily of triacylglycerols (TAG). TAG is accumulated in lipid droplets (LDs). Many small LDs are observed in the cell of the logarithmic phase. Later, a few big LDs are present in the cell in the TAG accumulating phase, suggesting a membranous fusion of LDs. Therefore, P. ellipsoidea is a viable algal strain with a potential for mass biodiesel production. The assessment of the environmental invasiveness of this strain is desirable prior to massive outdoor cultivation. P. ellipsoidea in an open pond spreads to the surrounding area by wind. In addition, nitrate, but not ammonia, is required for P. ellipsoidea to grow in mesotrophic environments.

Phosphorus is an essential element for life on the earth and is also crucial for modern agriculture, which is currently dependent on inorganic phosphate fertilizers produced from phosphate rock. Polyphosphate-accumulating organisms (PAOs) have been used for an application of biological phosphorus removal from wastewater. Chlorella is well-known trebouxiophycean green microalgae. When we observed Chlorella cells cultured under the sulfur-depleted condition using transmission electron microscopy, electron dense bodies (DBs) are found in the subcellular region. The commonly-used fluorescent dye, 4’, 6-diamidino-2-phenylindole (DAPI), can also be used for polyphosphate detection. Fluorescent microscopic observation shows that structure and subcellular distribution of the DBs are resemble with those of polyphosphate granules detected by DAPI. However, the relationship between polyphosphate, DBs and P-accumulation dynamics has been still unclear. Based on the energy dispersive X-ray analysis, the P signal is detected only in DBs. Molybdenum blue assay and 3D-TEM analysis shows that the DB is the site of polyphosphate accumulation during early and middle age of culture, indicating that Chlorella has a potential as a phosphate-accumulating organism.

Generally, eukaryotic cell has a set of organelles: three types of double-membrane-bound organelles (the cell nucleus, mitochondrion, and plastid), and four types of single-membrane-bound organelles [endoplasmic reticulum (ER), Golgi apparatus, peroxisome (microbody), and lysosome]. These organelles are essential for fulfilling the functions of eukaryotic cells. In textbooks, cell nuclei have been the focus of the study of cell proliferation, whereas division and proliferation of other six organelles have not been described much. This is because large number and complex shapes of these six organelles in cells of most of organisms from the amoeba to the higher animals and plants, making detailed structural study difficult. The primitive unicellular red alga Cyanidioschyzon merolae offers unique advantages for studying organelle division, because each cell contains a minimal set of basic eukaryotic organelles, the divisions of which occur in order and can be synchronized by light/dark cycles. In addition, the complete sequence of its genome has enabled proteomic analyses. As a result, we have discovered the plastid- (chloroplast) dividing apparatus (ring), the mitochondrion-dividing-apparatus (ring) and the peroxisome-dividing apparatus (ring), apparatuses that are essential for the survival of almost all the eukaryotic cells, ahead of the rest of the world, and then solved their structures and functions at the molecular level. This discovery also brought a new understanding of the birth of eukaryotic cells and organelles. The next work is to search smaller eukaryotic organisms to answer questions such as how such splitting devices were born, how much eukaryotes could be made smaller, and so on. We recently discovered the green alga Medakamo hakoo (Medakamo), which could possibly answer those questions. Here, I describe the research history of C. merolae as its foundation and discuss about future research of Medakamo.

Floral development is an important discipline within plant morphology, which in turn is the oldest botanical discipline. Recent achievements in molecular phylogeny, physiology and ecology have recalled the importance of floral development. To evaluate its contemporary relevance, we organized the JPR (Journal of Plant Research) symposium about the floral development. This symposium was co-organized by the Japanese Society of Plant Morphology and Fundación Flores (Chile).

Male flowers of Ceratophyllum demersum show a variable phyllotactic arrangement (merosity); spiral, trimerous, tetramerous and chaotic. We have demonstrated that, in a young primordium of male flower, both tepals and stamens show unidirectional initiation, first initiating on the abaxial side of the floral apex and only later on the adaxial side. In a flower at later developmental stage, inner stamens always show a spiral pattern. Our study indicated that the unidirectional initiation in early development is probably due to the pressure imposed by the leaf primordium at a higher node, and the spiral arrangement should be the original pattern in the stamen initiation. This result indicates similarities of the floral development in Ceratophyllum, at least in male flowers, with those of basal angiosperms except Nymphaeales. We conclude that mechanical forces on the adaxial side of the flower meristem are an important factor explaining the meristic variation not only in flowers of this species but also in flowers of all angiosperms. To examine this hypothesis, we have developed an experimental system using a micromanipulator with silicon device to produce an artificial mechanical force on the abaxial side of the young floral apex of Arabidopsis thaliana and to induce the development of flowers other than tetramerous ones. We have obtained some distorted flowers probably due to the mechanical force imposed by this device.

Fluorescent proteins enable us to visualize not only single cells but also organelles and molecules. However, plants have opaque bodies, complicated tissue structures containing air spaces, and autofluorescent compounds, which hinder fluorescence imaging of plants without mechanical sectioning. Recently, various clearing techniques have been developed for plant tissues to reduce the mismatch of refractive index and to remove the colored components. In this review, we will describe various clearing techniques for fluorescence imaging in plants.

Following the historical finding on the important contribution of endoreduplication on cell enlargement in the epidermis of leaves and hypocotyls, sometimes people misunderstand that cell size is always proportional to ploidy level. However, it was found that the impact of tetraploidization on cell size differs among cell types. More importantly, while endoreduplication occurs also in parenchymatous cells as well as epidermis in leaves, cell size of parenchymatous cells is very uniform. Our detailed analyses showed that the impact of endoreduplication on cell size is tissue-identity-dependent. Past reports on the relationship among changes in endoreduplication, cell size and organ size should be re-examined considering the above fact.

Eukaryotic cell possesses nucleus, endoplasmic reticulum, Golgi bodies, peroxisomes, mitochondria, and in case of plants, chloroplasts. The division and inheritance of all organelles is crucial for successful proliferation of eukaryotic cells. However, the molecular mechanisms of the division and inheritance of each organelle are largely unknown except for the nucleus because of the number of organelles that exist in the cell, the complex shapes that organelles exhibit, and the asynchrony of the timing of division for each organelle, in typical mammalian cultured cells and land plant cells. In contrast, unicellular red alga Cyanidioschyzon merolae is an extremely simple organism; C. merolae possesses a minimum set of organelles, which divide contemporarily in synchronous culture: the nuclear genome of C. merolae codes a small number of genes. In addition, transformation of C. merolae is feasible. Here I report the methods for producing stable transformants of C. merolae, the determination of cell-cycle stages by molecular markers, and construction of the gene expression profile as a platform for investigating organelle division and inheritance. I will also introduce my research regarding vacuolar inheritance revealed from the gene expression profile.

Chloroplasts (plastids) are able to synthesize energy-containing macromolecules for a great deal of living organisms. Consistent with their endosymbiotic origin, plastids maintain themselves by binary division. Plastid division is carried out by a ring complex called the plastid-dividing (PD) machinery; the PD machinery has inner and outer ring structures across the plastid membranes. Although many studies have been done to reveal the mechanisms of plastid division, much about the components and molecular mechanisms of the PD machinery remain to be discovered. My work demonstrated that: (1) the contractile force of the PD ring is generated via filament-sliding movement by dynamin proteins; (2) the PD ring is composed of polyglucan nanofilaments, synthesized by the glucosyltransferase PDR1; and (3) examination of the FtsZ ring reconstituted in a heterologous system revealed the assembly and contractile dynamics of the FtsZ ring. In addition, we have recently established isolation of the mitochondriondividing (MD) machinery and revealed that the ultrastructure and the dynamics of the isolated MD machinery were similar to those of the isolated PD machinery. Therefore, plastids and mitochondria divide by the action of supramolecular complexes “the PD and MD machineries” including dual contractible rings, the PD/MD ring and the FtsZ ring. These findings will lead to an understanding of how plastids and mitochondria were established during evolution.