Accumulation of Anthocyanins in Detached Leaves of Kalanchoë blossfeldiana: Relevance to the Effect of Methyl Jasmonate on This Process.

Accumulation of anthocyanins in detached leaves and in excised stems of Kalanchoë blossfeldiana kept under natural light conditions in the presence or absence of methyl jasmonate (JA-Me) was investigated. When the abaxial surface of detached leaves was held lower than the adaxial surface (the normal or natural position) under natural light conditions, anthocyanins were not accumulated on the abaxial side of the leaves. In contrast, when the adaxial surface of detached leaves was held lower than the abaxial surface (inverted position), anthocyanins were highly accumulated on the abaxial side of the leaves. These phenomena were independent of the growth stage of K. blossfeldiana as well as photoperiod. Application of JA-Me in lanolin paste significantly inhibited anthocyanin accumulation induced on the abaxial side of detached leaves held in an inverted position in a dose-dependent manner. Anthocyanin accumulation in the excised stem in response to natural light was also significantly inhibited by JA-Me in lanolin paste. Possible mechanisms of anthocyanin accumulation on the abaxial side of detached K. blossfeldiana leaves held in an inverted position under natural light conditions and the inhibitory effect of JA-Me on this process are described. The accompanying changes in the content of primary metabolites and histological analyses were also described.

Changes in Polar Metabolites Content during Natural and Methyl-Jasmonate-Promoted Senescence of Ginkgo biloba Leaves.

The present study clarified changes in the contents of polar metabolites (amino acids, organic acids, saccharides, cyclitols, and phosphoric acid) in leaf senescence in Ginkgo biloba with or without the application of methyl jasmonate (JA-Me) in comparison with those in naturally senescent leaf blades and petioles. The contents of most amino acids and citric and malic acids were significantly higher in abaxially, and that of myo-inositol was lower in abaxially JA-Me-treated leaves than in adaxially JA-Me-treated and naturally senescent leaves. The levels of succinic and fumaric acids in leaves treated adaxially substantially high, but not in naturally senescent leaves. In contrast, sucrose, glucose, and fructose contents were much lower in leaf blades and petioles treated abaxially with JA-Me than those treated adaxially. The levels of these saccharides were also lower compared with those in naturally senescent leaves. Shikimic acid and quinic acid were present at high levels in leaf blades and petioles of G. biloba. In leaves naturally senescent, their levels were higher compared to green leaves. The shikimic acid content was also higher in the organs of naturally yellow leaves than in those treated with JA-Me. These results strongly suggest that JA-Me applied abaxially significantly enhanced processes of primary metabolism during senescence of G. biloba compared with those applied adaxially. The changes in polar metabolites in relation to natural senescence were also discussed.

Mode of Action of 1-Naphthylphthalamic Acid in Conspicuous Local Stem Swelling of Succulent Plant, Bryophyllum calycinum: Relevance to the Aspects of Its Histological Observation and Comprehensive Analyses of Plant Hormones.

The mode of action of 1-naphthylphthalamic acid (NPA) to induce conspicuous local stem swelling in the area of its application to the growing internode in intact Bryophyllum calycinum was studied based on the aspects of histological observation and comprehensive analyses of plant hormones. Histological analyses revealed that NPA induced an increase in cell size and numerous cell divisions in the cortex and pith, respectively, compared to untreated stem. In the area of NPA application, vascular tissues had significantly wider cambial zones consisting of 5-6 cell layers, whereas phloem and xylem seemed not to be affected. This indicates that stem swelling in the area of NPA application is caused by stimulation of cell division and cell enlargement mainly in the cambial zone, cortex, and pith. Comprehensive analyses of plant hormones revealed that NPA substantially increased endogenous levels of indole-3-acetic acid (IAA) in the swelling area. NPA also increased endogenous levels of cytokinins, jasmonic acid, and its precursor, 12-oxo-phytodienoic acid, but did not increase abscisic acid and gibberellin levels. It was shown, using radiolabeled 14C-IAA, that NPA applied to the middle of internode segments had little effect on polar auxin transport, while 2,3,5-triiodobenzoic acid substantially inhibited it. These results strongly suggest that NPA induces changes in endogenous levels of plant hormones, such as IAA, cytokinins, and jasmonic acid, and their hormonal crosstalk results in a conspicuous local stem swelling. The possible different mode of action of NPA from other polar auxin transport inhibitors in succulent plants is extensively discussed.

Effect of Methyl Jasmonate on the Terpene Trilactones, Flavonoids, and Phenolic Acids in Ginkgo biloba L. Leaves: Relevance to Leaf Senescence.

The present study compared the effects of natural senescence and methyl jasmonate (JA-Me) treatment on the levels of terpene trilactones (TTLs; ginkgolides and bilobalide), phenolic acids, and flavonoids in the primary organs of Ginkgo biloba leaves, leaf blades, and petioles. Levels of the major TTLs, ginkgolides B and C, were significantly higher in the leaf blades of naturally senesced yellow leaves harvested on 20 October compared with green leaves harvested on 9 September. In petioles, a similar effect was found, although the levels of these compounds were almost half as high. These facts indicate the importance of the senescence process on TTL accumulation. Some flavonoids and phenolic acids also showed changes in content related to maturation or senescence. Generally, the application of JA-Me slightly but substantially increased the levels of TTLs in leaf blades irrespective of the difference in its application side on the leaves. Of the flavonoids analyzed, levels of quercetin, rutin, quercetin-4-glucoside, apigenin, and luteolin were dependent on the JA-Me application site, whereas levels of (+) catechin and (-) epicatechin were not. Application of JA-Me increased ferulic acid and p-coumaric acid esters in the petiole but decreased the levels of these compounds in the leaf blade. The content of p-coumaric acid glycosides and caffeic acid esters was only slightly modified by JA-Me. In general, JA-Me application affected leaf senescence by modifying the accumulation of ginkogolides, flavonoids, and phenolic acids. These effects were also found to be different in leaf blades and petioles. Based on JA-Me- and aging-related metabolic changes in endogenous levels of the secondary metabolites in G. biloba leaves, we discussed the results of study in the context of basic research and possible practical application.

Oxidative Cyclization of o-(1-Hydroxy-2-alkynyl)-N-tosylanilides for the Synthesis of 4-Quinolones.

The treatment of easily accessible o-(1-hydroxy-2-alkynyl)-N-tosylanilides 1 with excess manganese(IV) oxide in the presence of substoichiometric tetrabutylammonium iodide (TBAI) in chloroform (or in the absence of TBAI in dimethylformamide, DMF) promoted a sequential oxidation/intramolecular hydroamination to give 4-quinolones 3 and/or (Z)-2-alkylidene-3-oxindoles (Z)-4 in good yields. Possibly, MnO2 played dual roles as an oxidant and as a Lewis acidic activator of intermediary ynones 2. The product distributions between 3 and (Z)-4 could be controlled by the choice of solvents.

Formation of the Secondary Abscission Zone Induced by the Interaction of Methyl Jasmonate and Auxin in Bryophyllum calycinum: Relevance to Auxin Status and Histology.

The interaction of methyl jasmonate (JA-Me) and indole-3-acetic acid (IAA) to induce the formation of the secondary abscission zone in the middle of internode segments of Bryophyllum calycinum was investigated in relation to auxin status and histology. When IAA at 0.1% (w/w, in lanolin) was applied to the segments, the formation of the secondary abscission zone at a few mm above the treatment in the apical direction was observed. On the contrary, IAA at 0.5% (w/w, in lanolin) did not induce the formation of the secondary abscission zone. JA-Me at 0.5% (w/w, in lanolin) applied to the middle of internode segments kept in the normal (natural) or inverted positions also induced the formation of the secondary abscission zone below and above parts of the treatment. IAA at 0.5% applied to the cut surface of the upper part of the segments completely prevented the formation of the secondary abscission zone induced by JA-Me. Simultaneous application of IAA 0.5% with JA-Me 0.5% in the middle part of the internode segments induced the formation of the secondary abscission zone at 10 mm to 12 mm above the treatment. Histological analyses indicated that the formation of the secondary abscission zone was characterized by the presence of newly synthesized cell plates that resulted from periclinal cell division within one layer of mother cells in stems. The effects of IAA (0.1%) and JA-Me (0.5%) on the formation of the secondary abscission zone were histologically similar. Comprehensive analyses of plant hormones revealed that the balance of the endogenous levels of IAA in both sides adjacent to the abscission zone was significantly disturbed when the secondary abscission formation was induced by the application of IAA. These results strongly suggest that an auxin gradient is important in the formation of the secondary abscission zone in the internode segments of B. calycinum, and IAA gradient results from polar IAA transport from the application site. IAA is important in the regulation of formation of the secondary abscission zone induced by JA-Me. Further possible mechanisms of the formation of the secondary abscission zone in the internode segments of B. calycinum are also discussed in the interaction of JA-Me and IAA.

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.

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.

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.

A Possible Mode of Action of Methyl Jasmonate to Induce the Secondary Abscission Zone in Stems of Bryophyllum calycinum: Relevance to Plant Hormone Dynamics.

Plants can react to environmental stresses through the abscission of infected, damaged, or senescent organs. A possible mode of action of methyl jasmonate (JA-Me) to induce the formation of the secondary abscission zone (SAZ) in the stems of Bryophyllum calycinum was investigated concerning plant hormone dynamics. Internode segments were prepared mainly from the second or third internode from the top of plants with active elongation. JA-Me applied to the middle of internode segments induced the SAZ formation above and below the treatment after 5-7 days. At 6 to 7 days after JA-Me treatment, the above and below internode pieces adjacent to the SAZ were excised and subjected to comprehensive analyses of plant hormones. The endogenous levels of auxin-related compounds between both sides adjacent to the SAZ were quite different. No differences were observed in the level of jasmonic acid (JA), but the contents of 12-oxo-phytodienoic acid (OPDA), a precursor of JA, and N-jasmonyl-leucine (JA-Leu) substantially decreased on the JA-Me side. Almost no effects of JA-Me on the dynamics of other plant hormones (cytokinins, abscisic acid, and gibberellins) were observed. Similar JA-Me effects on plant hormones and morphology were observed in the last internode of the decapitated growing plants. These suggest that the application of JA-Me induces the SAZ in the internode of B. calycinum by affecting endogenous levels of auxin- and jasmonate-related compounds.

Gummosis in grape hyacinth (Muscari armeniacum) bulbs: hormonal regulation and chemical composition of gums.

The purpose of this study was to investigate the hormonal regulation of gummosis in grape hyacinth (Muscari armeniacum) bulbs, focusing especially on the chemical composition of the gums. The application of ethephon (2-chloroethylphosphonic acid), an ethylene-releasing compound, at 1% and 2% (w/w) in lanolin as well as ethylene induced gummosis in the bulbs within several days. Methyl jasmonate (JA-Me, 0.1-2% in lanolin) alone had no effect on gummosis. However, simultaneous application of JA-Me and ethephon led to extreme stimulation of ethephon-induced gummosis. Ethephon-induced gummosis in the bulbs depended on the maturation stage of the bulbs, increasing from April to July, but decreasing from August to September. Regardless of the presence of JA-Me, the application of ethephon to the inflorescence axis of grape hyacinths did not induce gummosis. Gel permeation chromatography analysis revealed that gums were homogenous polysaccharides with an average molecular mass of ca. 8.3 kDa. Analysis of the sugar composition of the gums after hydrolysis revealed that the molar ratio of Rha:Ara:Gal:GalA:GlcA was 25:10:40:7:15. These results suggest that principal factors of gummosis as well as the chemical composition of gums differ between species of bulbous plants.

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.

Correction: Cows painted with zebra-like striping can avoid biting fly attack.

[This corrects the article DOI: 10.1371/journal.pone.0223447.].

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.

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.

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.

Cows painted with zebra-like striping can avoid biting fly attack.

Experimental and comparative studies suggest that the striped coats of zebras can prevent biting fly attacks. Biting flies are serious pests of livestock that cause economic losses in animal production. We hypothesized that cows painted with black and white stripes on their body could avoid biting fly attacks and show fewer fly-repelling behaviors. Six Japanese Black cows were assigned to treatments using a 3 × 3 Latin-square design. The treatments were black-and-white painted stripes, black painted stripes, and no stripes (all-black body surface). Recorded fly-repelling behaviors were head throw, ear beat, leg stamp, skin twitch, and tail flick. Photo images of the right side of each cow were taken using a commercial digital camera after every observation and biting flies on the body and each leg were counted from the photo images. Here we show that the numbers of biting flies on Japanese Black cows painted with black-and-white stripes were significantly lower than those on non-painted cows and cows painted only with black stripes. The frequencies of fly-repelling behaviors in cows painted with black-and-white stripes were also lower than those in the non-painted and black-striped cows. These results thus suggest that painting black-and-white stripes on livestock such as cattle can prevent biting fly attacks and provide an alternative method of defending livestock against biting flies without using pesticides in animal production, thereby proposing a solution for the problem of pesticide resistance in the environment.

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.

Reaction of para-hydroxybenzoic acid esters with singlet oxygen in the presence of glutathione produces glutathione conjugates of hydroquinone, potent inducers of oxidative stress.

The determination and toxicological characterization of products of the reaction between p-hydroxybenzoic acid esters (parabens) and singlet oxygen ((1)O(2)) are very important because of the frequent use of parabens in cosmetics and possible generation of (1)O(2) in the skin. We observed (1)O(2)-dependent production of mono-, di-, and tri-substituted glutathione (GSH) conjugates of hydroquinone (HQ) during visible light-irradiation of a mixture of methyl or ethyl paraben and GSH in the presence of rose bengal (RB). 1,4-Benzoquinone (BQ) and HQ were produced during the irradiation in the absence of GSH. While a mixture of BQ and GSH produced only mono-substituted conjugate, irradiation of the mixture with RB produced mono-, di-, and tri-substituted conjugates. These observations indicate that (1)O(2) is involved both in the production of BQ and HQ from parabens and in the formation of multi-substituted GSH conjugates from mono-substituted conjugate. Tri-substituted conjugate generated larger amounts of hydrogen peroxide in an aqueous solution than mono-substituted conjugates or HQ did. Detection of semiquinone radical suggests that the autoxidation of conjugates is related to the generation of hydrogen peroxide. The results obtained in this study indicate that parabens may induce oxidative stress in the skin after conversion to GSH conjugates of HQ by reacting with (1)O(2) and GSH.

Kinetic study on ESR signal decay of nitroxyl radicals, potent redox probes for in vivo ESR spectroscopy, caused by reactive oxygen species.

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.