Prolonged exposure to hypergravity increases number and size of cells and enhances lignin deposition in the stem of Arabidopsis thaliana.

We have performed a lab-based hypergravity cultivation experiment using a centrifuge equipped with a lighting system and examined long-term effects of hypergravity on the development of the main axis of the Arabidopsis (Arabidopsis thaliana (L.) Heynh.) primary inflorescence, which comprises the rachis and peduncle, collectively referred to as the main stem for simplicity. Plants grown under 1 × g (gravitational acceleration on Earth) conditions for 20-23 days and having the first visible flower bud were exposed to hypergravity at 8 × g for 10 days. We analyzed the effect of prolonged hypergravity conditions on growth, lignin deposition, and tissue anatomy of the main stem. As a result, the length of the main stem decreased and cross-sectional area, dry mass per unit length, cell number, and lignin content of the main stem significantly increased under hypergravity. Lignin content in the rosette leaves also increased when they were exposed to hypergravity during their development. Except for interfascicular fibers, cross-sectional areas of the tissues composing the internode significantly increased under hypergravity in most types of the tissues in the basal part than the apical part of the main stem, indicating that the effect of hypergravity is more pronounced in the basal part than the apical part. The number of cells in the fascicular cambium and xylem significantly increased under hypergravity both in the apical and basal internodes of the main stem, indicating a possibility that hypergravity stimulates procambium activity to produce xylem element more than phloem element. The main stem was suggested to be strengthened through changes in its morphological characteristics as well as lignin deposition under prolonged hypergravity conditions.

How plants grow under gravity conditions besides 1 g: perspectives from hypergravity and space experiments that employ bryophytes as a model organism.

Plants have evolved and grown under the selection pressure of gravitational force at 1 g on Earth. In response to this selection pressure, plants have acquired gravitropism to sense gravity and change their growth direction. In addition, plants also adjust their morphogenesis in response to different gravitational forces in a phenomenon known as gravity resistance. However, the gravity resistance phenomenon in plants is poorly understood due to the prevalence of 1 g gravitational force on Earth: not only it is difficult to culture plants at gravity > 1 g(hypergravity) for a long period of time but it is also impossible to create a < 1 genvironment (µg, micro g) on Earth without specialized facilities. Despite these technical challenges, it is important to understand how plants grow in different gravity conditions in order to understand land plant adaptation to the 1 g environment or for outer space exploration. To address this, we have developed a centrifugal device for a prolonged duration of plant culture in hypergravity conditions, and a project to grow plants under the µg environment in the International Space Station is also underway. Our plant material of choice is Physcomitrium (Physcomitrella) patens, one of the pioneer plants on land and a model bryophyte often used in plant biology. In this review, we summarize our latest findings regarding P. patens growth response to hypergravity, with reference to our on-going "Space moss" project. In our ground-based hypergravity experiments, we analyzed the morphological and physiological changes and found unexpected increments of chloroplast size and photosynthesis rate, which might underlie the enhancement of growth and increase in the number of gametophores and rhizoids. We further discussed our approaches at the cellular level and compare the gravity resistance in mosses and that in angiosperms. Finally, we highlight the advantages and perspectives from the space experiments and conclude that research with bryophytes is beneficial to comprehensively and precisely understand gravitational responses in plants.

Functions and structure of roots and their contributions to salinity tolerance in plants.

Soil salinity is an increasing threat to the productivity of glycophytic crops worldwide. The root plays vital roles under various stress conditions, including salinity, as well as has diverse functions in non-stress soil environments. In this review, we focus on the essential functions of roots such as in ion homeostasis mediated by several different membrane transporters and signaling molecules under salinity stress and describe recent advances in the impacts of quantitative trait loci (QTLs) or genetic loci (and their causal genes, if applicable) on salinity tolerance. Furthermore, we introduce important literature for the development of barriers against the apoplastic flow of ions, including Na+, as well as for understanding the functions and components of the barrier structure under salinity stress.

Vegetative and reproductive growth of Arabidopsis under microgravity conditions in space.

We have performed a seed-to-seed experiment in the cell biology experiment facility (CBEF) installed in the Kibo (Japanese Experiment Module) in the International Space Station. The CBEF has a 1 × g compartment on a centrifuge and a microgravity compartment, to investigate the effects of microgravity on the vegetative and reproductive growth of Arabidopsis thaliana (L.) Heynh. Seeds germinated irrespective of gravitational conditions after water supply on board. Thereafter, seedlings developed rosette leaves. The time of bolting was slightly earlier under microgravity than under space 1 × g. Microgravity enhanced the growth rate of peduncles as compared with space 1 × g or ground control. Plants developed flowers, siliques and seeds, completing their entire life cycle during 62-days cultivation. Although the flowering time was not significantly affected under microgravity, the number of flowers in a bolted plant significantly increased under microgravity as compared with space 1 × g or ground control. Microscopic analysis of reproductive organs revealed that the longitudinal length of anthers was significantly shorter under microgravity when compared with space 1 × g, while the length of pistils and filaments was not influenced by the gravitational conditions. Seed mass significantly increased under microgravity when compared with space 1 × g. In addition, seeds produced in space were found not to germinate on the ground. These results indicate that microgravity significantly influenced the reproductive development of Arabidopsis plants even though Earth's gravitational environment is not absolutely necessary for them to complete their life cycle.

Correction to: A hypergravity environment increases chloroplast size, photosynthesis, and plant growth in the moss Physcomitrella patens.

The original article can be found online.

Plasma membrane-anchored chloroplasts are necessary for the gravisensing system of Ceratopteris richardii prothalli.

The prothalli of the fern Ceratopteris richardii exhibit negative gravitropism when grown in darkness. However, no sedimentable organelles or substances have been detected in the prothallial cells, suggesting that a non-sedimentable gravisensor exists. We investigated whether chloroplasts are involved in the gravisensing system of C. richardii prothalli. We used a clumped-chloroplast mutant, clumped chloroplast 1 (cp1), in which the chloroplasts are detached from the plasma membrane and clustered around the nucleus likely because of a partial deletion in the KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 gene. The cp1 mutation resulted in prothalli that had a significantly diminished gravitropic response, while the phototropic response occurred normally. These results suggest that plasma membrane-anchored chloroplasts in prothallial cells function as one of the gravisensors in C. richardii prothalli.

A hypergravity environment increases chloroplast size, photosynthesis, and plant growth in the moss Physcomitrella patens.

The physiological and anatomical responses of bryophytes to altered gravity conditions will provide crucial information for estimating how plant physiological traits have evolved to adapt to significant increases in the effects of gravity in land plant history. We quantified changes in plant growth and photosynthesis in the model plant of mosses, Physcomitrella patens, grown under a hypergravity environment for 25 days or 8 weeks using a custom-built centrifuge equipped with a lighting system. This is the first study to examine the response of bryophytes to hypergravity conditions. Canopy-based plant growth was significantly increased at 10×g, and was strongly affected by increases in plant numbers. Rhizoid lengths for individual gametophores were significantly increased at 10×g. Chloroplast diameters (major axis) and thicknesses (minor axis) in the leaves of P. patens were also increased at 10×g. The area-based photosynthesis rate of P. patens was also enhanced at 10×g. Increases in shoot numbers and chloroplast sizes may elevate the area-based photosynthesis rate under hypergravity conditions. We observed a decrease in leaf cell wall thickness under hypergravity conditions, which is in contrast to previous findings obtained using angiosperms. Since mosses including P. patens live in dense populations, an increase in canopy-based plant numbers may be effective to enhance the toughness of the population, and, thus, represents an effective adaptation strategy to a hypergravity environment for P. patens.

CT image-based 3D inflorescence estimation of Chrysanthemum seticuspe.

To study plant organs, it is necessary to investigate the three-dimensional (3D) structures of plants. In recent years, non-destructive measurements through computed tomography (CT) have been used to understand the 3D structures of plants. In this study, we use the Chrysanthemum seticuspe capitulum inflorescence as an example and focus on contact points between the receptacles and florets within the 3D capitulum inflorescence bud structure to investigate the 3D arrangement of the florets on the receptacle. To determine the 3D order of the contact points, we constructed slice images from the CT volume data and detected the receptacles and florets in the image. However, because each CT sample comprises hundreds of slice images to be processed and each C. seticuspe capitulum inflorescence comprises several florets, manually detecting the receptacles and florets is labor-intensive. Therefore, we propose an automatic contact point detection method based on CT slice images using image recognition techniques. The proposed method improves the accuracy of contact point detection using prior knowledge that contact points exist only around the receptacle. In addition, the integration of the detection results enables the estimation of the 3D position of the contact points. According to the experimental results, we confirmed that the proposed method can detect contacts on slice images with high accuracy and estimate their 3D positions through clustering. Additionally, the sample-independent experiments showed that the proposed method achieved the same detection accuracy as sample-dependent experiments.

Three-dimensional visualization of plant tissues and organs by X-ray micro-computed tomography.

Studies visualizing plant tissues and organs in three-dimension (3D) using micro-computed tomography (CT) published since approximately 2015 are reviewed. In this period, the number of publications in the field of plant sciences dealing with micro-CT has increased along with the development of high-performance lab-based micro-CT systems as well as the continuous development of cutting-edge technologies at synchrotron radiation facilities. The widespread use of commercially available lab-based micro-CT systems enabling phase-contrast imaging technique, which is suitable for the visualization of biological specimens composed of light elements, appears to have facilitated these studies. Unique features of the plant body, which are particularly utilized for the imaging of plant organs and tissues by micro-CT, are having functional air spaces and specialized cell walls, such as lignified ones. In this review, we briefly describe the basis of micro-CT technology first and then get down into details of its application in 3D visualization in plant sciences, which are categorized as follows: imaging of various organs, caryopses, seeds, other organs (reproductive organs, leaves, stems and petioles), various tissues (leaf venations, xylems, air-filled tissues, cell boundaries, cell walls), embolisms and root systems, hoping that wide users of microscopes and other imaging technologies will be interested also in micro-CT and obtain some hints for a deeper understanding of the structure of plant tissues and organs in 3D. Majority of the current morphological studies using micro-CT still appear to be at a qualitative level. Development of methodology for accurate 3D segmentation is needed for the transition of the studies from a qualitative level to a quantitative level in the future.

Demonstration of osmotically dependent promotion of aerenchyma formation at different levels in the primary roots of rice using a 'sandwich' method and X-ray computed tomography.

BACKGROUND AND AIMS: The effect of environmental factors on the regulation of aerenchyma formation in rice roots has been discussed for a long time, because aerenchyma is constitutively formed under aerated conditions. To elucidate this problem, a unique method has been developed that enables sensitive detection of differences in the development of aerenchyma under two different environmental conditions. The method is tested to determine whether aerenchyma development in rice roots is affected by osmotic stress. METHODS: To examine aerenchyma formation both with and without mannitol treatment in the same root, germinating rice (Oryza sativa) caryopses were sandwiched between two agar slabs, one of which contained 270 mm of mannitol. The roots were grown touching both slabs and were thereby exposed unilaterally to osmotic stress. As a non-invasive approach, refraction contrast X-ray computed tomography (CT) using a third-generation synchrotron facility, SPring-8 (Super photon ring 8 GeV, Japan Synchrotron Radiation Research Institute), was used to visualize the three-dimensional (3-D) intact structure of aerenchyma and its formation in situ in rice roots. The effects of unilateral mannitol treatment on the development of aerenchyma were quantitatively examined using conventional light microscopy. KEY RESULTS: Structural continuity of aerenchyma was clearly visualized in 3-D in the primary root of rice and in situ using X-ray CT. Light microscopy and X-ray CT showed that the development of aerenchyma was promoted on the mannitol-treated side of the root. Detailed light microscopic analysis of cross-sections cut along the root axis from the tip to the basal region demonstrated that aerenchyma developed significantly closer to the root tip on the mannitol-treated side of the root. CONCLUSIONS: Continuity of the aerenchyma along the rice root axis was morphologically demonstrated using X-ray CT. By using this 'sandwich' method it was shown that mannitol promoted aerenchyma formation in the primary roots of rice.

Three-dimensionally visualized rhizoid system of moss, Physcomitrium patens, by refraction-contrast X-ray micro-computed tomography.

Land plants have two types of shoot-supporting systems, root system and rhizoid system, in vascular plants and bryophytes. However, since the evolutionary origin of the systems is different, how much they exploit common systems or distinct systems to architect their structures is largely unknown. To understand the regulatory mechanism of how bryophytes architect the rhizoid system responding to environmental factors, we have developed the methodology to visualize and quantitatively analyze the rhizoid system of the moss, Physcomitrium patens, in 3D. The rhizoids having a diameter of 21.3 µm on the average were visualized by refraction-contrast X-ray micro-computed tomography using coherent X-ray optics available at synchrotron radiation facility SPring-8. Three types of shape (ring-shape, line and black circle) observed in tomographic slices of specimens embedded in paraffin were confirmed to be the rhizoids by optical and electron microscopy. Comprehensive automatic segmentation of the rhizoids, which appeared in three different form types in tomograms, was tested by a method using a Canny edge detector or machine learning. The accuracy of output images was evaluated by comparing with the manually segmented ground truth images using measures such as F1 score and Intersection over Union, revealing that the automatic segmentation using machine learning was more effective than that using the Canny edge detector. Thus, machine learning-based skeletonized 3D model revealed quite dense distribution of rhizoids. We successfully visualized the moss rhizoid system in 3D for the first time.

Development of the Casparian strip is delayed by blue light in pea stems.

To understand the regulatory mechanisms involved in tissue development by light, the kinetics of regulation of Casparian strip (CS) development in garden pea stems was studied. We found that short-term irradiation with white light delayed the development of the CS and used this delay to assess the quantitative effect of light on CS development. We examined the effect of the duration and fluence rates of white light treatment on CS development and observed a significant relationship between fluence and the delay in CS development indicating that the Bunsen-Roscoe law of reciprocity holds for this response. The effect of white light irradiation was not inhibited in the presence of a photosynthetic inhibitor, DCMU, or a carotenoid biosynthesis inhibitor, Norflurazon, indicating that the delay in CS development by light is a photomorphogenetic response rather than a subsidiary effect mediated by photosynthetic activity. An action spectrum for the response displayed a major peak in the blue-light region, suggesting a dominant role for blue-light receptors. A minor peak in the red-light region also suggested the possible involvement of phytochromes. Although phytochromes are known to contribute to blue-light responses, phytochrome-deficient mutants showed a normal delay of CS development in response to blue light, indicating that the response is not mediated by phytochrome and suggesting a role for one or more specific blue-light receptors.

Involvement of auxin dynamics in hypergravity-induced promotion of lignin-related gene expression in Arabidopsis inflorescence stems.

Recent studies have shown that hypergravity enhances lignification through up-regulation of the expression of lignin biosynthesis-related genes, although its hormonal signalling mechanism is unknown. The effects of hypergravity on auxin dynamics were examined using Arabidopsis plants that were transformed with the auxin reporter gene construct DR5::GUS. Hypergravity treatment at 300 g significantly increased ß-glucuronidase activity in inflorescence stems of DR5::GUS plants, indicating that endogenous auxin accumulation was enhanced by hypergravity treatment. The hypergravity-related increased expression levels of both DR5::GUS and lignin biosynthesis-related genes in inflorescence stems were suppressed after disbudding, indicating that the increased expression of lignin biosynthesis-related genes is dependent on an increase in auxin influx from the shoot apex.

A new galling insect model enhances photosynthetic activity in an obligate holoparasitic plant.

Insect-induced galls are microhabitats distinct from the outer environment that support inhabitants by providing improved nutrients, defence against enemies, and other unique features. It is intriguing as to how insects reprogram and modify plant morphogenesis. Because most of the gall systems are formed on trees, it is difficult to maintain them in laboratories and to comprehend the mechanisms operative in them through experimental manipulations. Herein, we propose a new model insect, Smicronyx madaranus, for studying the mechanisms of gall formation. This weevil forms spherical galls on the shoots of Cuscuta campestris, an obligate parasitic plant. We established a stable system for breeding and maintaining this ecologically intriguing insect in the laboratory, and succeeded in detailed analyses of the gall-forming behaviour, gall formation process, and histochemical and physiological features. Parasitic C. campestris depends on host plants for its nutrients, and usually shows low chlorophyll content and photosynthetic activity. We demonstrate that S. madaranus-induced galls have significantly increased CO2 absorbance. Moreover, chloroplasts and starch accumulated in gall tissues at locations inhabited by the weevil larvae. These results suggest that the gall-inducing weevils enhance the photosynthetic activity in C. campestris, and modify the plant tissue to a nutrient-rich shelter for them.

Visualization of Arabidopsis root system architecture in 3D by refraction-contrast X-ray micro-computed tomography.

Plant roots change their morphological traits in order to adapt themselves to different environmental conditions, resulting in the alteration of the root system architecture. To understand this mechanism, it is essential to visualize the morphology of the entire root system. To reveal effects of long-term alteration of gravity environment on root system development, we have performed an experiment in the International Space Station using Arabidopsis plants and obtained dried root systems grown in rockwool slabs. The X-ray computed tomography (CT) technique using industrial X-ray scanners has been introduced to visualize the root system architecture of crop species grown in soil in 3D non-invasively. In the case of the present study, however, the root system of Arabidopsis is composed of finer roots compared with typical crop plants and rockwool is also composed of fibers having similar dimension to that of the roots. A higher spatial resolution imaging method is required for distinguishing roots from rockwool. Therefore, in the present study, we tested refraction-contrast X-ray micro-CT using coherent X-ray optics available at the beamline of the synchrotron radiation facility SPring-8 for bio-imaging. We have found that a wide field of view but with low resolution obtained at the experimental Hutch 3 of this beamline provided an overview map of the root systems, while a narrow field of view but with high resolution obtained at the experimental Hutch 1 provided an extended architecture of the secondary roots, by a clear distinction between roots and individual rockwool fibers, resulting in the successful tracing of these roots from their basal regions.

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis.

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.

Use of ionic liquid for X-ray micro-CT specimen preparation of imbibed seeds.

X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.

[Effects of clinorotation on the structure of xylem in Arabidopsis roots].

Lignin, which is the most important component of vascular tissue, is essential material for the structural support of higher plants and considered to play a critical role in evolution of land plants. It has been postulated that development of secondary wall is mediated by gravity. However, effects of gravity on the development and the morphology of secondary cell wall have not been well investigated. In this study, effects of averaging of gravity vector using 2-D clinostat rotation on the structure of xylem was examined. The results indicated that the morphology of xylem is regulated by the orientation of gravity vector.

Effects of hypergravity environment on lignin formation in Arabidopsis.

Lignin and the secondary wall formation are essential for evolution of land plants. In this study, effects of hypergravity environment on the morphology of the secondary wall and the lignin content were examined in Arabidopsis thaliana. Xylem vessels showed intense staining with phloroglucinol-HCl and autofluorescence under UV light at the basal region of the flower stalk when seedlings grown for 3 days after hypergravity treatment for 24 hours. And, the flower stalk exposed to hypergravity showed slight increase in the lignin content. These results suggest that the lignin formation is positively regulated under hypergravity.