The gravity-induced re-localization of auxin efflux carrier cspin1 in cucumber seedlings: spaceflight experiments for immunohistochemical microscopy

ABSTRACT 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 1_g_ 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.

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ACID PROFILE, GROWTH PATTERN AND EXPRESSION OF _OSPIN_ GENES Article Open access 14 October 2020 INTRODUCTION Plants respond to gravity by changing their growth orientation and

morphology.1,2 The formation of a specialized protuberance, the peg, in cucurbitaceous seedlings is a unique gravimorphogenesis.2,3 When cucumber seeds (_Cucumis sativus_ L.) are placed in a

horizontal position and allowed to germinate, a peg forms on the lower side of the transition zone between the hypocotyl and the root. The peg anchors the lower seed coat in soil so that

the elongation of the hypocotyl pulls the cotyledons out of the seed coat. The peg therefore facilitates the emergence of seedlings from the hard seed coat. Cucumber seedlings have the

potential to develop a peg on each side of the transition zone as, when seeds are placed before germination in a vertical position with the radicles pointing down or under microgravity

conditions, a peg develops on each side.4 However, peg formation on the upper side of the transition zone is suppressed in response to gravity when the seedlings are grown in a horizontal

position on the ground.4 A phytohormone, auxin, has an important role in the lateral placement of peg formation in the transition zone.2,3 Application of indole-3-acetic acid (IAA), the main

auxin in plants, promotes peg development, and its endogenous concentration is significantly reduced in the peg-suppressed side (the upper side) of the transition zone.2,5,6 Furthermore,

treatment of seedlings with the auxin transport inhibitors 2,3,5-triiodobenzoic acid or 9-hydroxyfluorene-9-carboxylic acid blocks the suppression of peg formation on the upper side and

causes the development of a peg on each side of the transition zone, even when seedlings are germinated in a horizontal position.7 This suggests that gravity-modified transport of auxin is

required for the differential decrease in auxin level on the upper side of the transition zone in cucumber seedlings grown in a horizontal position. By contrast, the lower side of the

transition zone can maintain the higher auxin level required for peg formation.5,7 Plasma membrane-localized auxin efflux proteins of the PIN-FORMED (PIN) and P-glycoprotein families

facilitate the transport of auxin.8–10 In particular, the polarity of PIN localization corresponds to the direction of auxin transport.11,12 In Arabidopsis (_Arabidopsis thaliana_), AtPIN3

and AtPIN7 proteins that are expressed in gravisensing columella cells, respond to reorientation of roots by changing their localization to the side that has newly become the lower side of

the cells.13,14 Similarly, reorientation of Arabidopsis hypocotyls induces the re-localization of AtPIN3 to the lower side of gravisensing endodermal cells.15 In cucumber, we have shown that

reorientation of seedlings from a vertical position to a horizontal position induced changes in CsPIN1 localization in endodermis as well as asymmetric redistribution of auxin within 30 min

of reorientation in the transition zone.6 These observations led to the hypothesis that the change in CsPIN1 localization in the endodermis following the reorientation influences auxin

transport through the endodermis, which results in asymmetric auxin distribution in the transition zone.6 However, because these studies of CsPIN1 and AtPIN3 localization were studied using

longitudinal sections,6,13,15 the pathway of auxin transport via endodermal layers has been poorly understood not only in cucumber but also in other plant species including Arabidopsis.

Furthermore, the gravity-inducible change in PIN proteins remains to be verified in microgravity. Here we examined the CsPIN1 localization using the cross-sections of the transition zone of

cucumber seedlings grown under microgravity conditions. The results showed that endodermal cells re-localize CsPIN1 due to gravistimualtion and laterally transport auxin from the upper to

the lower flank, which explains the redistribution of auxin responsible for the lateral placement of peg formation in cucumber seedlings. RESULTS GROWTH AND MORPHOGENESIS OF CUCUMBER

SEEDLINGS IN MICROGRAVITY We previously reported that, when 24-h-old cucumber seedlings grown vertically were placed in a horizontal position, the localization of CsPIN1 in the endodermal

cell layer of the transition zone changed.6 We conducted spaceflight experiments on the International Space Station (ISS) to investigate the effects of gravistimulation on CsPIN1

localization. Before spaceflight experiments, we examined the effects of gravistimulation on peg formation using 24-h-old cucumber seedlings in ground control experiments. For this

experiments, we used a two-axis clinostat to rotate cucumber seedlings through three dimensions, because clinorotation randomizing the position of plants against gravity direction is used as

an analog for some plant responses under microgravity conditions.16–18 When cucumber seedlings were grown under clinorotated conditions for 24 h and then either maintained continuously on

the rotating clinostat or transferred to stationary conditions in a vertical position for 48 h, over half of the seedlings developed a peg on each side of the transition zone (Table 1). On

the other hand, when cucumber seedlings were clinorotated for 24 h after seed imbibition and then gravistimulated by placing them in a horizontal position, most seedlings developed a peg

only on the lower side of the transition zone after 48 h of growth (Table 1). It was shown that a peg formed on the new lower side but did not form on the new upper side when the

horizontally grown ~24-h-old cucumber seedlings were inverted up-side down.3 These results suggest that 24-h-old seedlings are still able to respond to gravity in determining the position of

peg formation. To examine the gravity-inducible re-localization of CsPIN1, therefore, we grew cucumber seedlings in space microgravity for 24 h and then exposed them to three different

gravitational conditions for 2 h as follows: (i) a continued microgravity environment, (ii) a 1_g_ centrifugal force in a longitudinal direction to the axis of hypocotyl–root of the

seedling, and (iii) a 1_g_ centrifugal force in a crosswise direction to the axis of hypocotyl–root of the seedling. The germination rate of cucumber seeds in our spaceflight experiments was

100% (Figure 1a–i). The roots of seedlings grown in microgravity grew in various directions slightly deviating from the seedling axis (Figure 1d,g). On the other hand, roots grew straight

and followed the direction of gravitational force when 1_g_ was applied longitudinally for 2 h (Figure 1h). Similarly, when seedlings were exposed to 1_g_ in the crosswise direction to the

axis of hypocotyl–root for 2 h their roots bent downward, due to the 1_g_ vector generated by centrifugation (Figure 1i). We compared the root lengths of seedlings grown in microgravity with

those of clinorotated seedlings on the ground. Although root length tended to be slightly longer in space, there was statistically no differences observed and there appeared to be

continuously growing for 2 h treatments after 24 h of germination (Figure 1j). EFFECT OF CENTRIFUGAL 1_G_ ON CSPIN1 LOCALIZATION IN CUCUMBER SEEDLINGS GROWN IN MICROGRAVITY We

immunohistochemically stained cross-sections of the transition zones of cucumber seedlings with anti-CsPIN1 antibodies. To characterize CsPIN1 localization in the endodermal cells of the

transition zone, we divided the endodermal cell layer, which was observed in half of the cross-section of the transition zone, into the three regions shown in Figure 2a,b. The cross-section

of the transition zone appeared elliptical in shape. The half of the transition zone of a non-gravistimulated seedling was further divided into halves (labeled ‘one side’ and ‘other side’)

by the major axis of the elliptical cross-section (Figure 2a). Both halves included endodermal cells situated in the ‘abaxial’ (in the plane of the cotyledon side) and ‘lateral’ (in the

plane of the non-cotyledon side) regions of the transition zone. Likewise, in the transition zone of seedlings exposed to 1_g_ centrifugal force applied in the crosswise direction to the

axis of hypocotyl–root, the endodermal cell layers were situated in both the ‘centripetal’ and ‘centrifugal’ regions divided by the major axis of the cross-section (Figure 2b). We further

categorized the types of endodermal cells with reference to the polarized localization of CsPIN1 signals within the cells. In the transition zones of non-gravistimulated seedlings grown

either in microgravity or exposed to 1_g_ centrifugal force applied in the longitudinal direction to the axis of hypocotyl–root (Figure 2a), CsPIN1 in type A endodermal cells localized to

the direction of the abaxial/one side; CsPIN1 in type B cells localized to the cell side parallel to the minor axis of the elliptic cross-section; and CsPIN1 in type C cells localized to the

direction of the abaxial/other side. Endodermal cells in which the CsPIN1 localization pattern was not distinct were classified as type D. The type D included cells that show CsPIN1 on all

around the plasma membrane or no CsPIN1 signals at all. In the transition zones of seedlings exposed to 1_g_ centrifugal force applied in the crosswise direction to the axis of

hypocotyl–root (Figure 2b), CsPIN1 in type A and C endodermal cells localized to the direction of the ‘centripetal’ and ‘centrifugal’ sides, respectively, and in type B cells CsPIN1

localized to the cell side parallel to the minor axis of the elliptic cross-section; again, cells other than types A, B, and C were classified as type D. When cucumber seedlings were grown

in microgravity for 24 h, CsPIN1 signals were detected at the cell side parallel to the minor axis of the cross-section of the transition zone (Figure 2c–f,k). When 24-h-old cucumber

seedlings grown in microgravity were incubated for a further 2 h in microgravity, the pattern of CsPIN1 in the abaxial sides but not in the lateral side slightly differed from that of

24-h-old cucumber seedlings grown in microgravity at _P_<0.05 level (Figure 2k). In cucumber seedlings grown for a further 2 h with 1_g_ centrifugal force applied in a longitudinal

direction to the axis of hypocotyl–root, the pattern of CsPIN1 in the abaxial side did not differ from that of 24-h-old cucumber seedlings grown in microgravity or that of the seedlings

grown for further 2 h in a vertical position (Figure 2k). However, when 24-h-old cucumber seedlings were exposed to 1_g_ centrifugal force applied in a crosswise direction to the axis of

hypocotyl–root for 2 h, the pattern of CsPIN1 localization in the abaxial/centripetal side and the lateral side significantly differed from that of 24-h-old cucumber seedlings grown in

microgravity at _P_<0.01 level (Figure 2g–j,k). This re-localization of CsPIN1 due to gravistimulation was much pronounced in the lateral side (Figure 2g,i,k). The localization pattern of

CsPIN1 in the transition zone in cucumber seedlings after exposing to 1_g_ centrifugal force for 2 h was characterized as an increased number of type C cells that localized CsPIN1 in

endodermal cells at the abaxial/centripetal and lateral sides (Figure 2g–i,k). In the abaxial/centrifugal side of the transition zone of cucumber seedlings exposed to 1_g_ centrifugal force

for 2 h, the pattern of CsPIN1 localization did not differ from that of 24-h-old cucumber seedlings grown in microgravity (Figure 2g,j,k). These results suggested that gravistimulation to

the space-grown cucumber seedlings with a 1_g_ centrifugal force applied in the crosswise direction induced a change in the localization of CsPIN1 and caused it to accumulate at the bottom

of the gravisensing endodermal cells in abaxial/centripetal and lateral sides of the transition zone. DISCUSSION Previously, we reported that CsPIN1 in the endodermal cells re-localize to

the bottom side upon reorientation of the seedlings, which is observable on the upper endodermal cells in the transition zone between the hypocotyl and the root of the horizontally placed

seedlings of cucumber.6 This result suggested that the pronounced auxin efflux due to CsPIN1 causes a decrease in auxin level in the upper side of the transition zone.6 Here our spaceflight

study demonstrate that CsPIN1 re-localization is a graviresponse, which could results in auxin redistribution in the transition zone of cucumber seedlings. In addition, the results of this

study reveal that endodermal cell layer with the polarized CsPIN1 localization could become a canal for the lateral auxin transport from the upper to the lower flank of the gravistimulated

transition zone. Auxin distribution in Arabidopsis is mainly regulated by the directional transport of auxin as follows: auxin synthesized in the shoot apex is transported toward the root

tip through vascular bundle cells by basipetally localized auxin efflux carriers, such as AtPIN1.11 Root columella cells expressing the auxin efflux carriers AtPIN3 and AtPIN7 transport

auxin toward the lateral root cap cells.13,14 Epidermal cells, which accumulate the AtPIN2 auxin efflux carrier, transport auxin from the lateral root caps to the elongation zone, in which

bending occurs due to a differential growth.12 The reorientation of roots from a vertical to a horizontal position induces the re-localization of AtPIN3 and AtPIN7 to the lower side of the

columella cells.13,14,19 As a result, auxin transport occurs asymmetrically from the columella cells to the lateral root cap cells and then to the lower flank of the elongation zone in a

horizontal position. This mechanism provides a good explanation for root gravitropism known as the Cholodny and Went hypothesis, which holds that gravitropic curvature of a growing plant

organ depends on asymmetric auxin distribution.20,21 In contrast to roots, which sense gravity via columella cells, lateral auxin transport in hypocotyls and shoots, in which endodermal

cells undergo gravistimulation, is poorly understood. Reorientation of cucumber seedlings from a vertical to a horizontal position increases CsPIN1 accumulation on the lower side of

endodermal cells in the upper endodermis of the transition zone,6 and in horizontally oriented hypocotyls of Arabidopsis, AtPIN3 accumulation decreases in the outer side of the upper

endodermal cells and in the inner side of the lower endodermal cells.15 This suggests that endodermal cells in the upper endodermis prevent auxin transport from vascular cells to cortical

cells in the transition zone of cucumber and in Arabidopsis hypocotyls. They could also promote auxin transport from the endodermis to vascular tissue in the upper flank of the

gravistimulated tissues. In the lower side of the gravistimulated hypocotyls of Arabidopsis, however, localization of AtPIN3 at the lower endodermal cells can facilitate transport of auxin

from endodermal cells to cortical cells.15 These models were based on the observation of the longitudinal sections and two-dimensional images of the transition zone and hypocotyls.6,15

However, the re-localization of auxin efflux carriers including AtPIN3 in endodermal layers in response to gravistimulation on the cross-sections has not been shown before. Our results using

cross-sections showed that, following horizontal placement of cucumber seedlings, the number of endodermal cells in which CsPIN1 was localized at the lower side of the cells in the upper

and lateral endodermis (Figure 2; abaxial/centripetal and lateral sides) of the transition zone increased. This observation implies that endodermal cells laterally transport auxin to enable

a greater accumulation of auxin in the lower flank of the transition zone. When cucumber seeds are placed and grown either in a vertical position or in microgravity conditions, a peg

develops on each side of the transition zone.4 We observed that, in the transition zone of cucumber seedlings grown in a vertical position or in microgravity, CsPIN1 signals in the abaxial

endodermal cells were detected at the adaxial side and the cell side parallel to the minor axis of the cross-section of the transition zone (Figure 3a). Under these growth conditions, a peg

develops on each side of the transition zone, and thus this pattern of CsPIN1 localization does not affect the symmetrical auxin distribution and cannot cause the reduction of auxin required

to suppress peg formation. This conclusion was consistent with that auxin-inducible _CsIAA1_ messenger RNA symmetrically accumulated in the transition zone of cucumber seedlings grown in

microgravity.5 These results of expression of auxin-inducible _CsIAA1_ gene, the localization of CsPIN1 proteins and peg formation suggest that the cucumber seedlings grown in a vertical

position and those grown in microgravity conditions possess physiologically a similar status. Recently, it has been described that the expression of auxin reporter gene (_pDR5r::GFP_) in

Arabidopsis grown in microgravity was identical to that of the ground control, although the expression of cytokinin reporter gene (_pARR5::GFP_) in microgravity differed from that on the

ground.22 Therefore, the responses of plants to 1_g_ would not affect auxin status but those to the direction of gravity would affect the PINs’ localization and then auxin distribution. The

present study suggests the endodermal cell layers of the transition zone become a canal for auxin transport from the upper to the lower side of the transition zone (Figure 3b). This lateral

auxin transport pathway composed of endodermal layers may induce an asymmetric distribution of auxin across the transition zone of cucumber seedlings. In addition, localization of CsPIN1 to

the lower side of endodermal cells on the upper side of the transition zone could contribute to the graviresponse negatively regulating peg formation by preventing auxin transport from

vascular tissue to the cortex and epidermis and/or by removing auxin from the cortex and epidermis. This model is probable, because asymmetric expression of an auxin-inducible gene,

_CsIAA1_, is detected in the epidermis and cortex across the transition zone.5,23,24 Regulation of auxin levels in the cortex and epidermis in this way may be an important factor responsible

for the lateral placement of peg formation in cucumber seedlings. In conclusion, our spaceflight experiments demonstrate that gravistimulation induces re-localization of CsPIN1 auxin efflux

carriers in endodermal cells in the transition zone of cucumber seedlings. Our results further suggest that re-localization of CsPIN1 auxin efflux carriers in endodermis due to

gravistimulation enables endodermal cell layers to transport auxin from the upper to the lower side of the transition zone of cucumber seedlings. MATERIALS AND METHODS PLANT MATERIALS AND

FIXATION On the ground, seven seeds of _Cucumis sativus_ L. cv. Shinfushinarijibai (Watanabe Seed Co., Kogota, Miyagi, Japan) were vertically or horizontally inserted into a crack within a

water-absorbent plastic foam placed in a plastic container, as shown in Supplementary Figure S1. A metallic pipe containing small holes and connected to a port for the injection of water was

inserted into the water-absorbent plastic foam (49×15×10 mm). The plastic containers holding the seeds, together with other instrumentation, were loaded into the STS-133 space shuttle,

Discovery, and launched from the Kennedy Space Center on 24 February 2011. While in orbit on the ISS, a plastic syringe fitted with a tap and a tube for drawing water from the water

reservoir was connected to the port of the plastic container. Pushing a plunger supplied 10 ml of distilled water to the foam so that imbibition of cucumber seeds was initiated. The plastic

container was placed in Measurement Experiment Unit B, which is fitted with a charge-coupled device camera, light-emitting diode lamps, and a temperature recorder. The Measurement Experiment

Unit B was then placed in the Cell Biology Experiment Facility, an incubator unit consisting of a microgravity compartment and a centrifuge compartment, which can provide centrifugal force

from 0.1 to 2.0 _g_. Cucumber seeds were allowed to germinate and the resulting seedlings were grown in microgravity or exposed to 1_g_ centrifugation at 25±1 °C in the dark. After

incubation, the seedlings were photographed and fixed with a fixative, acetic acid:ethanol:distilled water (5:63:32), using the Kennedy Space Center Fixation Tube (KFT). For fixation in

microgravity, each piece of water-absorbent plastic foam holding seven cucumber seedlings was detached from its container, put into the KFT containing the fixative, and stored at 4 °C in the

Minus Eighty Degree Celsius Laboratory Freezer for ISS until they were returned to Earth by the space shuttle _Atlantis_ (STS-135) and sent to our laboratory. The seedlings were kept

refrigerated at 4 °C during shipping and were stored in fixative for ~1 month. After the KFT was opened, the samples were infiltrated with newly prepared fixative, acetic acid: ethanol:

distilled water (5:63:32), and stored overnight at 4 °C. For ground experiments, the plastic containers were placed on the two-axis clinostat and three dimensionally rotated at 2 r.p.m. for

24 h.16–18 For gravistimulation of the seedlings, the containers were detached from the clinostat and placed on the ground ensuring that seedlings were placed either in a vertical or a

horizontal position for 2 h or 48 h. For non-gravistimulation, seedlings were further clinorotated for 2 h or 48 h. These experiments were performed at 25±1 °C in the dark. After incubation,

the samples were photographed. The length of roots of cucumber seedlings was measured using the photograph images and ImageJ ver. 1.42 software (NIH, Bethesda, MD, USA). IMMUNOHISTOCHEMICAL

ANALYSIS Histochemical staining for immunolocalization of CsPIN1 was performed as previously described,6 with some modifications. Although previously ethanol:chloroform:acetic acid (6:3:1)

was used as a fixative for immunohistochemical analysis of CsPIN1, this was changed to acetic acid:ethanol:distilled water (5:63:32) because of the safety regulations on the ISS. The

hypocotyl side of the transition zone contains four vascular strands and develops endodermal layers around each vascular strand whereas endodermal layers that surround two vascular strands

fuse in the root-side transition zone. CsPIN1 signals in seedlings fixed by the two fixatives were compared in advance: the signal intensity on the hypocotyl side of the transition zone was

much stronger in seedlings fixed with ethanol:chloroform:acetic acid (6:3:1) than in those fixed with acetic acid:ethanol:distilled water (5:63:32). CsPIN1 signal intensities on the root

side of the transition zone, however, were similar in seedlings fixed with the two fixatives. Thus, we analyzed the root side of the transition zone in this study. Fixed segments were used

for immunohistochemical analysis as described.6 To evaluate localization of CsPIN1 in endodermal cells of the transition zone, the types of endodermal cells were classified based on CsPIN1

localization in 6–10 images in half of the cross-section that were obtained from three to five cucumber seedlings (both halves of the cross-sections were used). The numbers of each cell type

were counted by single-blind assay as follows. The file names of images of immunohistochemistry were changed, and the changes were recorded. Then, one who did not know the change classified

the endodermal cells by CsPIN1 localization. After classification, the numbers of classified cells in each experiment were counted based on the records. STATISTICAL ANALYSIS Tukey’s method,

using KaleidaGraph Ver. 4.1J (Synergy Software, Reading, PA, USA), was adopted to analyze the root lengths of cucumber seedlings according to manufacturer’s instrument. For analyses of the

effects of gravistimulation and clinorotation on peg formation and of the effects of gravistimulation on CsPIN1 localization, Fisher’s exact (two-sided) test was performed using

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  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the ISS crew, Dr Satoshi Furukawa and his colleagues, for their in-orbit operations of our spaceflight experiments. We

are also grateful to all members of JAXA Flight Control Team for their preparation and ground operations of the spaceflight experiments. This study was conducted by the ‘Ground-based

Research Announcement for Space Utilization’ promoted by the Japan Space Forum. This work was also supported by a Grant-in-Aid for Scientific Research (B) (no. 020370017) from the Japan

Society for the Promotion of Science (JSPS), and a Grant-in-Aid for Scientific Research on Innovative Areas (no. 24620002) from the Ministry of Education, Culture, Sports, Science and

Technology of Japan, the Global COE program JO3 (Ecosystem Management Adapting to Global Changes) to H.T., by a Grant-in-Aid for Scientific Research (C) (K15K119130) from the JSPS to N.F.,

by the JSPS Research Fellowships for Young Scientists (K0219981) to C.Y., and by the Funding Program for Next-Generation World-Leading Researchers (GS002) to Y.M. AUTHOR INFORMATION AUTHORS

AND AFFILIATIONS * Graduate School of Life Sciences, Tohoku University, Sendai, Japan Chiaki Yamazaki, Nobuharu Fujii & Hideyuki Takahashi * Department of Science and Applications, Japan

Space Forum, Tokyo, Japan Chiaki Yamazaki, Toru Shimazu & Yasuo Fusejima * Faculty of Science, Yamagata University, Yamagata, Japan Yutaka Miyazawa * Future Development Division,

Advanced Engineering Services Co., Ltd, Tsukuba, Japan Motoshi Kamada * ISS Utilization and Operation Department, Japan Manned Space Systems Co., Tokyo, Japan Haruo Kasahara & Ikuko

Osada * JEM Utilization Center, Japan Aerospace Exploration Agency, Tsukuba, Japan Toru Shimazu & Akira Higashibata * Graduate School of Medicine, Teikyo University, Tokyo, Japan Takashi

Yamazaki * Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, Sagamihara, Japan Noriaki Ishioka Authors * Chiaki Yamazaki View author publications You can

also search for this author inPubMed Google Scholar * Nobuharu Fujii View author publications You can also search for this author inPubMed Google Scholar * Yutaka Miyazawa View author

publications You can also search for this author inPubMed Google Scholar * Motoshi Kamada View author publications You can also search for this author inPubMed Google Scholar * Haruo

Kasahara View author publications You can also search for this author inPubMed Google Scholar * Ikuko Osada View author publications You can also search for this author inPubMed Google

Scholar * Toru Shimazu View author publications You can also search for this author inPubMed Google Scholar * Yasuo Fusejima View author publications You can also search for this author

inPubMed Google Scholar * Akira Higashibata View author publications You can also search for this author inPubMed Google Scholar * Takashi Yamazaki View author publications You can also

search for this author inPubMed Google Scholar * Noriaki Ishioka View author publications You can also search for this author inPubMed Google Scholar * Hideyuki Takahashi View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS C.Y., N.F., Y.M., H.T., M.K., H.K., I.O., T.S., Y.F., A.H., T.Y. and N.I. designed the research and

performed the experiments. C.Y., N.F., and H.T. analyzed the data and wrote the paper. All authors read, reviewed, and approved the manuscript. CORRESPONDING AUTHORS Correspondence to

Nobuharu Fujii or Hideyuki Takahashi. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Supplementary Information accompanies the

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Yamazaki, C., Fujii, N., Miyazawa, Y. _et al._ The gravity-induced re-localization of auxin efflux carrier CsPIN1 in cucumber seedlings: spaceflight experiments for immunohistochemical

microscopy. _npj Microgravity_ 2, 16030 (2016). https://doi.org/10.1038/npjmgrav.2016.30 Download citation * Received: 11 April 2016 * Revised: 17 June 2016 * Accepted: 17 July 2016 *

Published: 15 September 2016 * DOI: https://doi.org/10.1038/npjmgrav.2016.30 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link

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