A symposium entitled “Imaging and its related technologies in plant biology” was held at the 75^th meeting of the Botanic Society of Japan in September 2011. This symposium was co-supported by the Japanese Society of Plant Morphology and by the Non-profit Organization Integrated Imaging Research Support (NPO IIRS). In this issue, researchers who contributed the symposium write how imaging and its related technologies had and have been contributed to the study of plant biology.

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Results obtained from physiological analyses of constitutively observed cytoplasmic streaming in characean internodal cells and light-induced cytoplasmic streaming in Vallisneria mesophyll cells done by the centrifuge microscope of stroboscopic type were briefly summarized, touching recently revealed responsible motor proteins and endoplasmic reticulum structures. Special emphasis was placed on the putative cross bridges formed upon the cessation of cytoplasmic streaming in characean cells and on the mechanical properties of endoplasm.

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It is not easy to record the local changes of cytoplasmic structure and the global cellular event simultaneously. We have developed a microscope system, Global-Local Live Imaging Microscope (GLIM) System, that can record the local molecular structural dynamics and the whole cell event in parallel without exchanging objectives. Here, we explain the GLIM system, and demonstrate some results in plant cell division and morphogenesis. The possible applications of GLIM system in future will also be discussed.

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Intracellular distribution of chloroplasts is regulated by light and the motile system of chloroplast relocation movement generally depends on actin filaments. The basic mechanism, however, has not been explored. We examined the dynamic behavior of actin filaments during chloroplast movement in transformed plants expressing GFP-mouse talin. Using custom made fluorescent time-lapse imaging system equipped with a microbeam irradiator, we found short actin filaments exhibiting rapid turnover along the chloroplast periphery, termed as chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments showed biased localiztion on the leading edge of chloroplasts during chloroplast movement and the biased localization was cancelled in conjunction with chloroplast anchoring. In Arabidopsis thaliana, no apparent cp-actin filaments formation was observed in chloroplast unusual positioning 1 (chup1) mutant plants that is impaired in a gene encoding an actin-binding protein. Furthermore, cp-actin filaments and their biased localization during chloroplast movement were observed in protonema cells of Physcomitrella patens. These data suggest that plants evolved a unique actin-based motile system for chloroplast movement.

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Gravity is a ubiquitous force on the earth and a crucial environmental signal for living organisms. To adapt and survive in the gravitational field, plants sense the gravity vector (magnitude and direction) and change their morphology accordingly. These are widespread phenomena known as ‘gravity resistance’ and ‘gravitropism’. We have studied the early process of shoot gravitropism, gravity sensing, using molecular genetic techniques in combination with two novel microscopes, a vertical-stage confocal microscope and a centrifuge microscope. The vertical-stage confocal microscope is a vertically oriented microscope equipped with a rotatable stage, a spinning-disk confocal scanning unit and a back-illuminated EM-CCD camera, which allows fluorescence imaging of the Arabidopsis stem specimen before and after gravistimulation (changes in the direction of gravity). The centrifuge microscope is a spinning upright microscope equipped with a radio system, which allows bright-field imaging during centrifugation (changes in the magnitude of gravity). In this review, we will introduce the recently developed microscopes that are essential to gain new insights into gravity sensing mechanisms in Arabidopsis inflorescence stems.

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Microinjection has been widely used by researches in the cell biology field. However, conventional microinjection methods are not suitable for plant cells because the plant cells have high turgor pressure which often causes influx of the cytoplasm into a glass needle. This phenomenon results in needle clogging. Our novel microinjection method, laser-assisted thermal-expansion microinjection (LTM) can generate higher pressure by thermal expansion of laser absorbent. By LTM method, we can inject materials into not only plant cells but also various types targets including budding yeast, nuclei and organelles without clogging a needle. In this review article, we summarize the principle and applications of LTM method.

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Land plants include ca. 270,000 species which occupy various habitats throughout the world. Unique and “funny” morphologies, which are suitable for their survival, have been evolved in the Land plants. Following featured articles review how such “funny” morphologies have been evolved. They also show the future direction of morphological studies on non-model plants.

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Epiphytes are plants that live on other plant bodies and account for 10% of vascular plants worldwide. We suggested that the obligate epiphyte life-form evolved from climber or secondary hemiepiphyte life-form in Davalliaceae and related ferns, based on molecular phylogeny and comparative morphology. Several morphological characters, long-creeping rhizomes and peltate scales seem likely to play significant roles in the hypothesized evolution. An anomalous morphology was observed in the secondary hemiepiphytic Oleandra pistillaris. The internodes are variously long and sometimes to 2 m long, resulting in an irregular phyllotaxy. Observation of the rhizomes showed that the species is characterized by the rhizome dimorphism; the rhizomes are erect and creeping. The erect rhizome produces leaves in apparent whorls separated by subequal internodes, but the creeping rhizome is dorsiventral with variously long internodes. Another secondary hemiepiphytic Nephrolepis also has similar dimorphic rhizomes. It is in contrast to the general monomorphic rhizome with regular phyllotaxy in other life forms of the related ferns.

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Plants grow indeterminately by the activities of shoot and root apical meristems at the opposite ends of poles of the embryo, establishing a vertical body plan. By contrast, aquatic eudicot family Podostemaceae develop a horizontal plant body that allows them to adapt to their peculiar habitats, i.e., submerged rock-surfaces in fast-flowing rivers. Seedlings of Terniopsis species, belonging to subfamily Tristichoideae, retain a vertical growth by shoot and root meristems, while members of subfamily Podostemoideae cease vertical growth by rudimentary embryonic shoot and root meristems. Instead, an adventitious root arises from the lateral side of the hypocotyl and form adventitious shoots on the dorsal or lateral side. Here I review our recent study focusing on the loss/reduction of the embryonic shoot and root meristems. Comparative embryonic anatomical studies revealed that the developmental changes in the cellular embryogenesis of Podostemoideae caused the loss of embryonic shoot and root meristems.

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Osmunda lancea is the rheophyte which is considered to have speciated from O. japonica and has an adaptive morphology, stenophylly. In order to clarify the genetic architecture of stenophylly, using the spores collected from a putative F1 hybrid, F2 hybrids were synthesized. The six leaf characters were measured in the F2 hybrids with ternate compound leaves. As the results, the angle of pinna base is significantly correlated to the number of veinlets in most basal basiscopic vein and that in most basal acroscopic vein, and the correlation coefficients were 0.520 and 0.426, respectively. Based on the results of molecular marker analyses, a locus appeared to be associated with the angle of pinna base, but this locus was not associated with the other measured characters.

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The leaves of some plant species are able to change their morphology in response to environmental conditions. This phenomenon is termed heterophylly. Various aquatic plants exhibit drastic changes in leaf shape in response to submerged aquatic conditions. Heterophyllic variation ranges from mere modification of leaf width to drastic alteration in the outline of leaves and is interpreted as an adaptation to aquatic habitats. Although this phenomenon is widely observed among angiosperms, there is limited information on the regulation of heterophyllic switch in leaf development. Here, we have reviewed existing knowledge on leaf development and heterophylly and have introduced Neobeckia aquatica as an emerging model to elucidate the mechanisms underlying heterophylly.

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A new type of actin rods formed in both of the nucleus and the cytoplasm are implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. The rods are composed of hexagonally cross-linked actin tubules with 13 nm diameter, and include S-adenosyl-L-homocysteine hydrolase. In addition to actin rods, the complex of G-actin and fat droplets are formed in the spore cytoplasm. Half of the actin molecules in the spores are tyrosine-phosphorylated. The high level of the phosphorylation is required for stabilizing spores. D-glucose is a trigger molecule for the actin dephosphorylation. In the contact-sensitive plant Mimosa pudica L., actin of the main pulvinus is heavily tyrosine-phosphorylated and changes in the extent of phosphorylation correlate with the degree of bending of the plant’s petioles. Also in dormant forms of the true slime mold Physarum polycephalum actin is phosphorylated. In D. discoideum, SrfA, a homologous gene to the MADS-box family, is involved in the formation of the actin rods as well as actin phosphorylation.

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Podostemaceae (riverweeds) is a family of aquatic angiosperms growing on rocks under running water. The plants evolved unique body plans such as root-borne shoots for adaptation to such an extreme habitat. The basal subfamilies Tristichoideae and Weddellinoideae, like most other angiosperms, have typical shoot apical meristems (SAMs). The subfamily Podostemoideae, however, is devoid of SAM and form new leaves from the meristematic basal region of preexisting leaves. A recent gene expression analysis found that the SAM-less Podostemoideae leaf has mixed nature of SAM and leaf and provided an insight into the evolution of shoot in Podostemaceae.

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For energy, food, and oxygen, almost all organisms depend on photosynthesis by chloroplasts (plastids), which proliferate by division. In chloroplast division, the plastid-dividing (PD) ring is a main structure of the PD machinery and is a universal structure in the plant kingdom. However, the components and formation of the PD ring have been enigmatic. By proteomic analysis of PD machineries isolated from Cyanidioschyzon merolae, we identified the glycosyltransferase protein plastid-dividing ring 1 (PDR1), which constructs the PD ring and is widely conserved from red alga to land plants. Electron microscopy showed that the PDR1 protein forms a ring with carbohydrates at the chloroplast-division site. Fluorometric saccharide ingredient analysis of purified PD ring filaments showed that only glucose was included. Thus, the chloroplasts are divided by the PD ring, which is a bundle of PDR1-mediated polyglucan filaments.

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In most flowering plants, the apical-basal body axis is initiated by an asymmetric division of the polarized zygote. This division generates 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. Despite the obvious importance, how the zygote polarizes and how this asymmetry is translated to the embryo axis have been obscure. Recently we identified that Arabidopsis zinc-finger transcription factor WRKY2 regulates both of the zygote polarity and the embryo patterning. In this review, we summarize how we found WRKY2 and discuss what we should do to reveal the molecular mechanism of WRKY2-dependent axis formation.

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Double fertilization is a one of the remarkable features in angiosperms. A pollen grain contains two sperm cells. One of the two sperm cells fertilizes the egg cell to produce embryo. Another sperm cell fertilizes the central cell to produce an endosperm which supplies nutrient to the embryo. The movement of the sperm cells during double fertilization was never caught before. Therefore, basic questions for double fertilization, such as what are paths of sperm cells to reach target female gametes and why two sperm cells can fertilize different partners are not resolved. In this decade, technical innovation of live-cell imaging has enabled to reveal the sperm cell movement during double fertilization. In this review, we discuss the double fertilization process from pollen tube discharge to gamete fusion, focusing on sperm cell movement by live-cell imaging and molecular genetic approaches.

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Vacuoles have pivotal roles in intracellular digestion, pH and ion homeostasis, and metabolite storage. Although vacuoles can be synthesized de novo (Hoh et al. 1995, Catlett and Weisman 2000), they are inherited from mother cells to daughter cells during cell division. This vacuolar inheritance would enable cells to function properly right after mitosis. Yeast vacuoles are inherited using class V myosin and actin. In unicellular red alga Cyanidioschyzon merolae, which lacks conventional actin and myosin, vacuoles are inherited by binding to mitochondria. In this review, I summarize our current knowledge in mechanisms for vacuole inheritance and discuss future perspectives.

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Maize (Zea mays L.) is one of the most representative C₄ plants. However, several reports have suggested the occurrence of C₃ photosynthesis in this C₄ plant. We examined the photosynthetic characteristics of the coleoptiles, as well as the first, the second, and the third leaves of maize seedlings at their maturities. All the foliage leaves examined, including the first leaf sheath, exhibited representative Kranz anatomy, low CO₂ compensation point, and expression of genes for enzymes involved in the C₄-dicarboxylic acid cycle. In contrast, coleoptiles showed no Kranz anatomy and extremely high CO₂ compensation point. The expression of C₄-specific genes in the coleoptiles was hampered at various levels. These results strongly suggest that the maize coleoptiles do not perform typical C₄ photosynthesis.

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