Effect of microgravity on mammalian embryo development evaluated at the International Space Station.

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

Comprehensive analyses of plant hormones in etiolated pea and maize seedlings grown under microgravity conditions in space: Relevance to the International Space Station experiment "Auxin Transport".

Functional relationships between endogenous levels of plant hormones in the growth and development of shoots in etiolated Alaska pea and etiolated Golden Cross Bantam maize seedlings under different gravities were investigated in the "Auxin Transport" experiment aboard the International Space Station (ISS). Comprehensive analyses of 31 species of plant hormones of pea and maize seedlings grown under microgravity (µg) in space and 1 g conditions were conducted. Principal component analysis (PCA) and a multiple regression analysis with the dataset from the plant hormone analysis of the etiolated pea seedlings grown under µg and 1 g conditions in the presence and absence of 2,3,5-triiodobenzoic acid (TIBA) revealed endogenous levels of auxin correlated positively with bending and length of epicotyls. Endogenous cytokinins correlated negatively with them. These results suggest an interaction of auxin and cytokinins in automorphogenesis and growth inhibition of etiolated Alaska pea epicotyls grown under µg conditions in space. Less polar auxin transport with reduced endogenous levels of auxin increased endogenous levels of cytokinins, resulting in changing the growth direction of epicotyls and inhibiting growth. On the other hand, almost no close relationship between endogenous plant hormone levels and growth and development in etiolated maize seedlings grown was observed under µg conditions in space, as per Schulze et al. (1992). However, endogenous levels of IAA in the seedlings grown under µg conditions in space were significantly higher than those grown on Earth, similar to the cases of polar auxin transport already reported.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station.

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

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.

Suppression of Cortical Microtubule Reorientation and Stimulation of Cell Elongation in Arabidopsis Hypocotyls under Microgravity Conditions in Space.

How microgravity in space influences plant cell growth is an important issue for plant cell biology as well as space biology. We investigated the role of cortical microtubules in the stimulation of elongation growth in Arabidopsis (Arabidopsis thaliana) hypocotyls under microgravity conditions with the Resist Tubule space experiment. The epidermal cells in the lower half of the hypocotyls of wild-type Columbia were longer in microgravity than at on-orbit 1 g, which precipitated an increase in the entire hypocotyl length. In the apical region, cortical microtubules adjacent to the outer tangential wall were predominantly transverse to the long axis of the cell, whereas longitudinal microtubules were predominant in the basal region. In the 9th to 12th epidermal cells (1 to 3 mm) from the tip, where the modification of microtubule orientation from transverse to longitudinal directions (reorientation) occurred, cells with transverse microtubules increased, whereas those with longitudinal microtubules decreased in microgravity, and the average angle with respect to the transverse cell axis decreased, indicating that the reorientation was suppressed in microgravity. The expression of tubulin genes was suppressed in microgravity. These results suggest that under microgravity conditions, the expression of genes related to microtubule formation was downregulated, which may cause the suppression of microtubule reorientation from transverse to longitudinal directions, thereby stimulating cell elongation in Arabidopsis hypocotyls.

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.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth.

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.

Microarray profile of gene expression in etiolated Pisum sativum seedlings grown under microgravity conditions in space: Relevance to the International Space Station experiment "Auxin Transport".

This paper introduces the use of microarray data technology with Medicago (Medicago truncatula) microarrays to characterize global changes in the transcript abundance of etiolated Alaska pea (Pisum sativum L.) seedlings grown under microgravity (µg) conditions in comparison with those under artificial 1 g conditions on the International Space Station. Of the 44,000 genes of the Medicago microarray platform, more than 25,000 transcripts of pea seedlings were hybridized, suggesting that the microarray platform for Medicago could be useful in the study of gene expression of etiolated pea seedlings grown under µg conditions in space. Gene array data were analyzed according to stringent criteria that restricted the scored genes for specific hybridization values at least twofold. Expression of 1362 and 1558 genes in proximal side (the proximal side) and distal side of the epicotyl to the cotyledons (the distal side), respectively, were highly affected by µg conditions in space. Of the genes analyzed, 407 of 1362 transcripts in the proximal side and 740 of 1558 transcripts in the distal side were expressed at ratios at least twofold. However, in the presence of the auxin transport inhibitor TIBA, 212 of 399 transcripts and 255 of 477 transcripts were expressed at ratios at least twofold as high in the proximal and the distal sides of epicotyls in the seedlings grown under µg conditions, respectively. Based on Venn diagram analysis, 31 transcripts and 24 transcripts were found to commonly increase and decrease, respectively, under µg conditions in space. Venn analysis revealed six auxin-related genes and three water channel AQUAPORIN genes that were responsive to gravity. Among 6 auxin-related genes, the accumulation of transcripts of Auxin-induced protein 5NG4 and Indole-3-acetic acid-amido synthetase GH3.3 tended to increase, and that of Auxin-induced protein, Auxin response factor, SAUR-like auxin-responsive family protein and Auxin response factor tended to decrease under µg conditions, whereas there were no statistic differences between under µg and artificial 1 g conditions. Similarly there were no statistic differences between under µg conditions and artificial 1 g, but the accumulation of NIP3-1 and Plasma membrane intrinsic protein11, and AQUAPORIN1/Tonoplast intrinsic protein tended to increase and decrease, respectively. A possible role of auxin-related genes and AQUAPORIN genes in regulating growth of etiolated pea seedlings grown under µg conditions in space is discussed.

Altered localisation of ZmPIN1a proteins in plasma membranes responsible for enhanced-polar auxin transport in etiolated maize seedlings under microgravity conditions in space.

In the International Space Station experiment 'Auxin Transport', polar auxin transport (PAT) in shoots of etiolated maize (Zea mays L. cv. Golden Cross Bantam) grown under microgravity in space was substantially enhanced compared with those grown on Earth. To clarify the mechanism, the effects of microgravity on expression of ZmPIN1a encoding essential auxin efflux carrier and cellular localisation of its products were investigated. The amounts of ZmPIN1a mRNA in the coleoptiles and the mesocotyls in space-grown seedlings were almost the same as those in 1 g-grown seedlings, but its products were not. Immunohistochemical analysis with anti-ZmPIN1a antibody revealed a majority of ZmPIN1a localised in the basal side of plasma membranes of endodermal cells in the coleoptiles and the mesocotyls, and in the basal and lateral sides of plasma membranes in coleoptile parenchymatous cells, in which it directed towards the radial direction, but not towards the vascular bundle direction. Microgravity dramatically altered ZmPIN1a localisation in plasma membranes in coleoptile parenchymatous cells, shifting mainly towards the vascular bundle direction. These results suggest that mechanism of microgravity-enhanced PAT in maize shoots is more likely to be due to the enhanced ZmPIN1a accumulation and the altered ZmPIN1a localisation in parenchymatous cells of the coleoptiles.

Characterization of amyloid ß fibril formation under microgravity conditions.

Amyloid fibrils are self-assembled and ordered proteinaceous supramolecules structurally characterized by the cross-ß spine. Amyloid formation is known to be related to various diseases typified by neurogenerative disorders and involved in a variety of functional roles. Whereas common mechanisms for amyloid formation have been postulated across diverse systems, the mesoscopic morphology of the fibrils is significantly affected by the type of solution condition in which it grows. Amyloid formation is also thought to share a phenomenological similarity with protein crystallization. Although many studies have demonstrated the effect of gravity on protein crystallization, its effect on amyloid formation has not been reported. In this study, we conducted an experiment at the International Space Station (ISS) to characterize fibril formation of 40-residue amyloid ß (Aß(1-40)) under microgravity conditions. Our comparative analyses revealed that the Aß(1-40) fibrilization progresses much more slowly on the ISS than on the ground, similarly to protein crystallization. Furthermore, microgravity promoted the formation of distinct morphologies of Aß(1-40) fibrils. Our findings demonstrate that the ISS provides an ideal experimental environment for detailed investigations of amyloid formation mechanisms by eliminating the conventionally uncontrollable factors derived from gravity.

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.

Microgravity Affects the Level of Matrix Polysaccharide 1,3:1,4-ß-Glucans in Cell Walls of Rice Shoots by Increasing the Expression Level of a Gene Involved in Their Breakdown.

The plant cell wall provides each cell with structural support and mechanical strength, and thus, it plays an important role in supporting the plant body against the gravitational force. We investigated the effects of microgravity on the composition of cell wall polysaccharides and on the expression levels of genes involved in cell wall metabolism using rice shoots cultivated under artificial 1 g and microgravity conditions on the International Space Station. The bulk amount of the cell wall obtained from microgravity-grown shoots was comparable with that from 1 g-grown shoots. However, the analysis of sugar constituents of matrix polysaccharides showed that microgravity specifically reduced the amount of glucose (Glc)-containing polysaccharides such as 1,3:1,4-ß-glucans, in shoot cell walls. The expression level of a gene for endo-1,3:1,4-ß-glucanase, which hydrolyzes 1,3:1,4-ß-glucans, largely increased under microgravity conditions. However, the expression levels of genes involved in the biosynthesis of 1,3:1,4-ß-glucans were almost the same under both gravity conditions. On the contrary, microgravity scarcely affected the level and the metabolism of arabinoxylans. These results suggest that a microgravity environment promotes the breakdown of 1,3:1,4-ß-glucans, which, in turn, causes the reduced level of these polysaccharides in growing rice shoots. Changes in 1,3:1,4-ß-glucan level may be involved in the modification of mechanical properties of cell walls under microgravity conditions in space.

Microbial Monitoring in the International Space Station and Its Application on Earth.

The space habitat is a confined environment with a simple ecosystem that consists mainly of microorganisms and humans. Changes in the pathogenicity and virulence of bacteria, as well as in astronauts' immune systems, during spaceflight may pose potential hazards to crew health. To ensure microbiological safety in the space habitat, a comprehensive analysis of environmental microbiota is needed to understand the overall microbial world in this habitat. The resulting data contribute to evidence-based microbial monitoring, and continuous microbial monitoring will provide information regarding changes in bioburden and microbial ecosystem; this information is indispensable for microbiological management. Importantly, the majority of microbes in the environment are difficult to culture under conventional culture conditions. To improve understanding of the microbial community in the space habitat, culture-independent approaches are required. Furthermore, there is a need to assess the bioburden and physiological activity of microbes during future long-term space habitation, so that the "alert" and/or "action" level can be assessed based on real-time changes in the microbial ecosystem. Here, we review the microbial monitoring in the International Space Station-Kibo, and discuss how these results will be adapted to the microbial control in space habitation and pharmaceutical and food processing industries.

Gravity-regulated localization of PsPIN1 is important for polar auxin transport in etiolated pea seedlings: Relevance to the International Space Station experiment.

To clarify the mechanism of gravity-controlled polar auxin transport, we conducted the International Space Station (ISS) experiment "Auxin Transport" (identified by NASA's operation nomenclature) in 2016 and 2017, focusing on the expression of genes related to auxin efflux carrier protein PsPIN1 and its localization in the hook and epicotyl cells of etiolated Alaska pea seedlings grown for three days in the dark under microgravity (µg) and artificial 1 g conditions on a centrifuge in the Cell Biology Experiment Facility (CBEF) in the ISS, and under 1 g conditions on Earth. Regardless of gravity conditions, the accumulation of PsPIN1 mRNA in the proximal side of epicotyls of the seedlings was not different, but tended to be slightly higher as compared with that in the distal side. 2,3,5-Triiodobenzoic acid (TIBA) also did not affect the accumulation of PsPIN1 mRNA in the proximal and distal sides of epicotyls. However, in the apical hook region, TIBA increased the accumulation of PsPIN1 mRNA under µg conditions as compared with that under artificial 1 g conditions in the ISS. The accumulation of PsPIN1 proteins in epicotyls determined by western blotting was almost parallel to that of mRNA of PsPIN1. Immunohistochemical analysis with a specific polyclonal antibody of PsPIN1 revealed that a majority of PsPIN1 in the apical hook and subapical regions of the seedlings grown under artificial 1 g conditions in the ISS localized in the basal side (rootward) of the plasma membrane of the endodermal tissues. Conversely, in the seedlings grown under µg conditions, localization of PsPIN1 was greatly disarrayed. TIBA substantially altered the cellular localization pattern of PsPIN1, especially under µg conditions. These results strongly suggest that the mechanisms by which gravity controls polar auxin transport are more likely to be due to the membrane localization of PsPIN1. This physiologically valuable report describes a close relationship between gravity-controlled polar auxin transport and the localization of auxin efflux carrier PsPIN1 in etiolated pea seedlings based on the µg experiment conducted in space.

Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Polar auxin transport is essential to maintain growth and development of etiolated pea and maize seedlings grown under 1 g conditions: Relevance to the international space station experiment.

We conducted "Auxin Transport" space experiments in 2016 and 2017 in the Japanese Experiment Module (JEM) on the International Space Station (ISS), with the principal objective being integrated analyses of the growth and development of etiolated pea (Pisum sativum L. cv Alaska) and maize (Zea mays L. cv Golden Cross Bantam) seedlings under true microgravity conditions in space relative to auxin dynamics. Etiolated pea seedlings grown under microgravity conditions in space for 3 days showed automorphogenesis. Epicotyls and roots bent ca. 45° and 20° toward the direction away from the cotyledons, respectively, whereas those grown under artificial 1 g conditions produced by a centrifuge in the Cell Biology Experimental Facility (CBEF) in space showed negative and positive gravitropic response in epicotyls and in roots, respectively. On the other hand, the coleoptiles of 4-day-old etiolated maize seedlings grew almost straight, but the mesocotyls curved and grew toward a random direction under microgravity conditions in space. In contrast, the coleoptiles and mesocotyls of etiolated maize seedlings grown under 1 g conditions on Earth were almost straight and grew upward or toward the direction against the gravity vector. The polar auxin transport activity in etiolated pea epicotyls and in maize shoots was significantly inhibited and enhanced, respectively, under microgravity conditions in space as compared with artificial 1 g conditions in space or 1 g conditions on Earth. An inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA) substantially affected the growth direction and polar auxin transport activity in etiolated pea seedlings grown under both artificial 1 g and microgravity conditions in space. These results strongly suggest that adequate polar auxin transport is essential for gravitropic response in plants. Possible mechanisms enhancing polar auxin transport in etiolated maize seedlings grown under microgravity conditions in space are also proposed.

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space-grown seedlings.

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space-grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild-type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation-like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild-type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge-generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92-100% under both microgravity and 1 g conditions. LED-synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild-type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Root-tip-mediated inhibition of hydrotropism is accompanied with the suppression of asymmetric expression of auxin-inducible genes in response to moisture gradients in cucumber roots.

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots.