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.

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.

Understanding the Mechanisms of Gravity Resistance in Plants.

To understand gravity resistance in plants, it is necessary to analyze the changes induced when the magnitude of gravity in a growth environment is modified. Microgravity in space provides appropriate conditions for analyzing gravity resistance mechanisms. Experiments carried out in space involve a large number of constraints and are quite different from ground-based experiments. Here, we describe basic procedures for space-based experiments to study gravity resistance in plants. An appropriate cultivation chamber must be selected according to the growing period of the plants and the purpose of the experiment. After cultivation, the plant material is fixed with suitable fixatives in appropriate sample storage containers such as the Chemical Fixation Bag. The material is then analyzed with a variety of methods, depending on the purpose of the experiment. Plant material fixed with the RNAlater® solution can be used sequentially to determine the mechanical properties of the cell wall, RNA extraction (which is necessary for gene-expression analysis), estimate the enzyme activity of cell wall proteins, and measure the levels and compositions of cell wall polysaccharides. The plant material can also be used directly for microscopic observation of cellular components such as cortical microtubules.

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.

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.

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.

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.

Procedures for chemical fixation in immunohistochemical analyses of PIN proteins regulating polar auxin transport: Relevance to spaceflight experiments.

The mechanism by which gravity controls the polar transport of auxin, a plant hormone regulating multiple physiological processes in higher plants, remains unclear, although an important role of PIN proteins as efflux carriers/facilitators in polar auxin transport is suggested. We are going to study the effect of microgravity on the polar transport of auxin, focusing on the cellular localization of its efflux carrier, PsPIN1 in etiolated pea seedlings and ZmPIN1a in etiolated maize seedlings grown under microgravity conditions on the International Space Station (ISS) using immunohistochemical analyses according to space experimental plans (Ueda, 2016). To obtain adequate results regarding the cellular localization of functional proteins, prolonged chemical fixation processes as well as chemical fixatives should be well-matched to the properties of functional proteins as antigens since experimental analyses will be performed on the ground after keeping samples for a long duration on the ISS. As a result of ground verification, clear detection of the cellular localization of PsPIN1 and ZmPIN1a immunohistochemically was successful based on the results of several kinds of chemical fixation tested, even when etiolated pea and maize seedlings were fixed by immersion in chemical fixative for a long duration. The addition of 0.1% (w/v) Nonidet P-40 to chemical fixative composed of 50% (v/v) ethanol and 5% (v/v) acetic acid or that of 50% (v/v) methanol and 5% (v/v) acetic acid has led to a significant improvement in the immunohistochemical detection of PsPIN1 or ZmPIN1a. These chemical fixatives were also shown to be storage-stable for a long time before use. In this study, adequate chemical fixatives and fixation protocols were developed, which can be used to detect localization of PsPIN1 and ZmPIN1a proteins in young etiolated pea and maize seedlings, respectively, using anti PsPIN1 and ZmPIN1a antibodies. These protocols can be used in spaceflight experiments to investigate the effects of the microgravity environment on the ISS on PIN protein localization in pea and maize seedlings.

Regulation of asymmetric polar auxin transport by PsPIN1 in endodermal tissues of etiolated Pisum sativum epicotyls: focus on immunohistochemical analyses.

This manuscript reports the production of specific polyclonal antibodies for PsPIN1, a putative auxin efflux carrier in Alaska pea (Pisum sativum L.) plants, and the cellular immunolocalization of PsPIN1. When pea seeds were set with the seed axis horizontal to the upper surface of a rockwool block, and allowed to germinate and grow for 3 days in the dark, the epicotyl grew upward. On the other hand, the application of 2,3,5-triiodobenzoic acid (TIBA) inhibited graviresponse. In the subapical epicotyl regions, PsPIN1 has been found to localize in the basal side of the plasma membrane of cells in endodermal tissues. Asymmetric PsPIN1 localization between the proximal and distal sides of the epicotyl was observed, the total amounts of PsPIN1 being more abundant in the proximal side. The asymmetric PsPIN1 distribution between the proximal and distal sides of the epicotyl was well correlated with unequal polar auxin transport as well as asymmetric accumulation of mRNA of PsPIN1 (Ueda et al. in Biol Sci Space 26:32-41, 2012; Ueda et al. in Plant Biol 16(suppl 1):43-49, 2014). In the proximal side of an apical hook, PsPIN1 localized in the basal side of the plasma membrane of cells in endodermal tissues, whereas in the distal side, the abundant distribution of PsPIN1 localized in the basal-lower (endodermal) side of the basal plasma membrane, suggesting possible lateral auxin movement from the distal side to the proximal side in this region. The application of TIBA significantly reduced the amount of PsPIN1 in the proximal side of epicotyls, but little in the distal side. These results suggest that unequal auxin transport in epicotyls during the early growth stage of etiolated pea seedlings is derived from asymmetric PsPIN1 localization in the apical hook and subapical region of epicotyls, and that asymmetric transport between the proximal and distal sides of epicotyls is required for the graviresponse of epicotyls.

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.

Modification of growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls grown under microgravity conditions in space.

We carried out a space experiment, denoted as Aniso Tubule, to examine the effects of microgravity on the growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls, using lines in which microtubules are visualized by labeling tubulin or microtubule-associated proteins (MAPs) with green fluorescent protein (GFP). In all lines, GFP-tubulin6 (TUB6)-, basic proline-rich protein1 (BPP1)-GFP- and spira1-like3 (SP1L3)-GFP-expressing using a constitutive promoter, and spiral2 (SPR2)-GFP- and GFP-65 kDa MAP-1 (MAP65-1)-expressing using a native promoter, the length of hypocotyls grown under microgravity conditions in space was longer than that grown at 1 g conditions on the ground. In contrast, the diameter of hypocotyls grown under microgravity conditions was smaller than that of the hypocotyls grown at 1 g. The percentage of cells with transverse microtubules was increased under microgravity conditions, irrespective of the lines. Also, the average angle of the microtubules with respect to the transverse cell axis was decreased in hypocotyls grown under microgravity conditions. When GFP fluorescence was quantified in hypocotyls of GFP-MAP65-1 and SPR2-GFP lines, microgravity increased the levels of MAP65-1, which appears to be involved in the maintenance of transverse microtubule orientation. However, the levels of SPR2 under microgravity conditions were comparable to those at 1 g. These results suggest that the microgravity-induced increase in the levels of MAP65-1 is involved in increase in the transverse microtubules, which may lead to modification of growth anisotropy, thereby developing longer and thinner hypocotyls under microgravity conditions in space.

Gravitropism interferes with hydrotropism via counteracting auxin dynamics in cucumber roots: clinorotation and spaceflight experiments.

Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (µG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under µG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and µG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in µG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.

Persistence of plant hormone levels in rice shoots grown under microgravity conditions in space: its relationship to maintenance of shoot growth.

We investigated the effects of microgravity environment on growth and plant hormone levels in dark-grown rice shoots cultivated in artificial 1 g and microgravity conditions on the International Space Station (ISS). Growth of microgravity-grown shoots was comparable to that of 1 g-grown shoots. Endogenous levels of indole-3-acetic acid (IAA) in shoots remained constant, while those of abscisic acid (ABA), jasmonic acid (JA), cytokinins (CKs) and gibberellins (GAs) decreased during the cultivation period under both conditions. The levels of auxin, ABA, JA, CKs and GAs in rice shoots grown under microgravity conditions were comparable to those under 1 g conditions. These results suggest microgravity environment in space had minimal impact on levels of these plant hormones in rice shoots, which may be the cause of the persistence of normal growth of shoots under microgravity conditions. Concerning ethylene, the expression level of a gene for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, the key enzyme in ethylene biosynthesis, was reduced under microgravity conditions, suggesting that microgravity may affect the ethylene production. Therefore, ethylene production may be responsive to alterations of the gravitational force.

The gravity-induced re-localization of auxin efflux carrier CsPIN1 in cucumber seedlings: spaceflight experiments for immunohistochemical microscopy.

Reorientation of cucumber seedlings induces re-localization of CsPIN1 auxin efflux carriers in endodermal cells of the transition zone between hypocotyl and roots. This study examined whether the re-localization of CsPIN1 was due to the graviresponse. Immunohistochemical analysis indicated that, when cucumber seedlings were grown entirely under microgravity conditions in space, CsPIN1 in endodermal cells was mainly localized to the cell side parallel to the minor axis of the elliptic cross-section of the transition zone. However, when cucumber seeds were germinated in microgravity for 24 h and then exposed to 1g centrifugation in a direction crosswise to the seedling axis for 2 h in space, CsPIN1 was re-localized to the bottom of endodermal cells of the transition zone. These results reveal that the localization of CsPIN1 in endodermal cells changes in response to gravity. Furthermore, our results suggest that the endodermal cell layer becomes a canal by which auxin is laterally transported from the upper to the lower flank in response to gravity. The graviresponse-regulated re-localization of CsPIN1 could be responsible for the decrease in auxin level, and thus for the suppression of peg formation, on the upper side of the transition zone in horizontally placed seedlings of cucumber.

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space.

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.

Gravistimulation changes the accumulation pattern of the CsPIN1 auxin efflux facilitator in the endodermis of the transition zone in cucumber seedlings.

Cucumber (Cucumis sativus) seedlings grown in a horizontal position develop a specialized protuberance (or peg) on the lower side of the transition zone between the hypocotyl and the root. This occurs by suppressing peg formation on the upper side via a decrease in auxin resulting from a gravitational response. However, the gravity-stimulated mechanism of inducing asymmetric auxin distribution in the transition zone is poorly understood. The gravity-sensing tissue responsible for regulating auxin distribution in the transition zone is thought to be the endodermal cell. To characterize the gravity-stimulated mechanism, the auxin efflux facilitator PIN-FORMED1 (CsPIN1) in the endodermis was identified and the localization of CsPIN1 proteins during the gravimorphogenesis of cucumber seedlings was examined. Immunohistochemical analysis revealed that the accumulation pattern of CsPIN1 protein in the endodermal cells of the transition zone of cucumber seedlings grown horizontally differed from that of plants grown vertically. Gravistimulation for 30 min prompted changes in the accumulation pattern of CsPIN1 protein in the endodermis as well as the asymmetric distribution of auxin in the transition zone. Furthermore, 2,3,5-triiodobenzoic acid inhibited the differential distribution of auxin as well as changes in the accumulation pattern of CsPIN1 in the endodermis of the transition zone during gravistimulation. These results suggest that the altered pattern of CsPIN1 accumulation in the endodermis in response to gravistimulation influences lateral auxin transport through the endodermis, resulting in asymmetric auxin distribution in the transition zone.

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus.

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.