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.

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.

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.

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.

Breakdown of middle lamella pectin by (●) OH during rapid abscission in Azolla.

Azolla, a small water fern, abscises its roots and branches within 30 min upon treatment with various stresses. This study was conducted to test whether, in the rapid abscission that occurs in Azolla, breakdown of wall components of abscission zone cells by (●) OH is involved. Experimentally generated (●) OH caused the rapid separation of abscission zone cells from detached roots and the rapid shedding of roots from whole plants. Electron microscopic observations revealed that (●) OH rapidly and selectively dissolved a well-developed middle lamella between abscission zone cells and resultantly caused rapid cell separation and shedding. Treatment of abscission zones of Impatiens leaf petiole with (●) OH also accelerated the separation of abscission zone cells. However, compared with that of Azolla roots, accelerative effects in Impatiens were weak. A large amount of (●) OH was cytochemically detected in abscission zone cells both of Azolla roots and of Impatiens leaf petioles. These results suggest that (●) OH is involved in the cell separation process not only in the rapid abscission in Azolla but also in the abscission of Impatiens. However, for rapid abscission to occur, a well-developed middle lamella, a unique structure, which is sensitive to the attack of (●) OH, might be needed.

Analysis of apical hook formation in Alaska pea with a 3-D clinostat and agravitropic mutant ageotropum.

The formation of the apical hook in dicotyledonous seedlings is believed to be effected by gravity in the dark. However, this notion is mostly based on experiments with the hook formed on the hypocotyl, and no detailed studies are available with the developmental manners of the hook, particularly of the epicotyl hook. The present study aims at clarifying the dynamics of hook formation including the possible involvement of gravity. Time-course studies with normal Alaska pea (Pisum sativum L., cv. Alaska) and an agravitropic pea mutant, ageotropum, under the 1-g conditions and on a 3-D clinostat revealed that (1) the apical hook of the epicotyl forms by the development of the arc-shaped plumule of the embryo existing in the non-germinated seed. The process of formation consists of two stages: development and partial opening, which are controlled by some intrinsic property of the plumule, but not gravity. Approximately when the epicotyl emerges from the seed coat, the hook is established in both pea varieties. In Alaska the established hook is sustained or enhanced by gravity, resulting in a delay of hook opening compared with on a clinostat, which might give an incorrect idea that gravity causes hook formation. (2) During the hook development and opening processes the original plumular arc holds its orientation unchanged to be an established hook, which, therefore, is at the same side of the epicotyl axis as the cotyledons. This is true for both Alaska and ageotropum under 1-g conditions as well as on the clinostat, supporting finding (1). (3) Application of auxin polar transport inhibitors, hydroxyfluorenecarboxylic acid, naphthylphthalamic acid, and triiodobenzoic acid, suppressed the curvature of hook by equal extents in Alaska as well as ageotropum, suggesting that the hook development involves auxin polar transport probably asymmetrically distributed across the plumular axis by some intrinsic property of the plumule not directly related with gravity action.

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH.

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0-5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.

Inhibition of nitrogen-fixing activity of the cyanobiont affects the localization of glutamine synthetase in hair cells of Azolla.

In the Azolla-Anabaena association, the host plant Azolla efficiently incorporates and assimilates ammonium ions that are released from the nitrogen-fixing cyanobiont, probably via glutamine synthetase (GS; EC 6.3.1.2) in hair cells, which are specialized cells protruding into the leaf cavity. In order to clarify the regulatory mechanism underlying ammonium assimilation in the Azolla-Anabaena association, Azolla plants were grown under an argon environment (Ar), in which the nitrogen-fixing activity of the cyanobiont was inhibited specifically and completely. The localization of GS in hair cells was determined by immunoelectron microscopy and quantitative analysis of immunogold labeling. Azolla plants grew healthily under Ar when nitrogen sources, such as NO(3)(-) and NH(4)(+), were provided in the growth medium. Both the number of cyanobacterial cells per leaf and the heterocyst frequency of the plants under Ar were similar to those of plants in a nitrogen environment (N(2)). In hair cells of plants grown under Ar, regardless of the type of nitrogen source provided, only weak labeling of GS was observed in the cytoplasm and in chloroplasts. In contrast, in hair cells of plants grown under N(2), abundant labeling of GS was observed in both sites. These findings indicate that specific inhibition of the nitrogen-fixing activity of the cyanobiont affects the localization of GS isoenzymes. Ammonium fixed and released by the cyanobiont could stimulate GS synthesis in hair cells. Simultaneously, the abundant GS, probably GS1, in these cells, could assimilate ammonium rapidly.

Localization of glutamine synthetase isoforms in hair cells of Azolla leaves.

Immunoelectron microscopy and a quantitative analysis of immunogold labeling of a glutamine synthetase (GS; EC 6.3.1.2) revealed that, in mesophyll cells of mature leaves of Azolla filiculoides, almost all GS was present in chloroplasts. By contrast, in hair cells, abundant labeling of GS was observed both in chloroplasts and in the cytoplasm. In hair cells of cyanobiont-free plants, the labeling of GS of both chloroplasts and cytoplasm was very weak compared to that of cyanobiont-containing plants. The findings suggest that hair cells play an important role in the assimilation of ammonia released by the cyanobiont.

Purification and properties of a carbonyl reductase involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate from Cylindrocarpon sclerotigenum IFO 31855.

A NADPH-dependent carbonyl reductase (CSCR1) was purified to homogeneity from Cylindrocarpon sclerotigenum IFO 31855. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a >99% enantiomer excess. The relative molecular mass of the enzyme was estimated to be 68,000 by gel filtration chromatography and 24,800 on SDS polyacrylamide gel electrophoresis. The enzyme had an extremely narrow substrate specificity and it highly reduced conjugated diketone, 2,3-butanedion, in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by HgCl(2) (100%), 5,5'-dithiobis(2-nitrobenzoic acid) (56%), dicoumarol (42%), and CuSO(4) (46%). The N-terminal amino acid sequence of the enzyme (P-Q-G-I-P-T-A-S-R-L) showed no apparent similarity with those of other oxidoreductases.