François Bizet

Veronica Pereda-Loth

Hugo Chauvet

Joëlle Gérard

Brigitte Eche

Christine Girousse

Monique Courtade

Gérald Perbal

Valérie Legué
email: valerie.legue@uca.fr

Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity

Gravity is a permanent environmental signal guiding plant growth and development. Gravity sensing in plants starts with the displacement of starch-filled plastids called statoliths, ultimately leading to auxin redistribution and organ curvature. While the involvement in gravity sensing of several actors such as calcium is known, the effect of statolith displacement on calcium changes remains enigmatic. Microgravity is a unique environmental condition offering the opportunity to decipher this link. In this study, roots of Brassica napus were grown aboard the International Space Station (ISS) either in microgravity or in a centrifuge simulating Earth gravity. The impact of short simulated gravity onset and removal was measured on statolith positioning and intracellular free calcium was assessed using pyroantimonate precipitates as cytosolic calcium markers. Our findings show that a ten-minute onset or removal of gravity induces very low statolith displacement, but which is, nevertheless, associated with an increase of the number of pyroantimonate precipitates. These results highlight that a change in the cytosolic calcium distribution is triggered in absence of a significant statolith displacement.

Gravity sensing in plants occurs in specialized cells called statocytes which are located in the shoot endodermis and in the central root cap [START_REF] Morita | Directional Gravity Sensing in Gravitropism[END_REF] . Statocytes contain starch-filled plastids called statoliths. Being denser than the cytoplasm, the statolith position depends on the direction of gravity. Thus, it is assumed that statocytes constitute one of the plant gravity sensors in the starch-statolith hypothesis [START_REF] Sack | Plant gravity sensing[END_REF][START_REF] Driss-Ecole | A polarized cell: the root statocyte[END_REF] . In roots, statocytes show an intriguing polarization of organelles. The nucleus is located at the shootward end of the cell whereas the endoplasmic reticulum (ER) is mostly at the rootward end [START_REF] Driss-Ecole | A polarized cell: the root statocyte[END_REF] . The ER has long been suspected to be involved in the early gravity transduction pathway. In the 70 s, longitudinal sections of root apices highlighted compression of the rootward ER complex by sedimented statoliths [START_REF] Sievers | Ultrastructure of gravity-perceiving cells in plant roots[END_REF][START_REF] Sievers | Verursacht differentieller Druck der Amyloplasten auf ein komplexes Endomembransystem die Geoperzeption in Wurzeln?[END_REF][START_REF] Volkmann | Amyloplasten und Endomembranen: das Geoperzeptionsystem der Primärwurzel[END_REF] . This observation that the weight of statoliths is sufficient to locally deform the ER membranes was later confirmed using high-resolution electron tomography [START_REF] Leitz | Statolith sedimentation kinetics and force transduction to the cortical endoplasmic reticulum in gravity-sensing Arabidopsis columella cells[END_REF] . Accordingly, the ability of plants to orient their growth along the gravity vector, called gravitropism, was hypothesized to be the consequence of the pressure exerted by statoliths on the rootward ER complex. This widespread hypothesis was used to explain differences in root sensitivity to gravity observed at various inclination changes [START_REF] Iversen | The starch-statolith hypothesis and the interaction of amyloplasts and endoplasmic reticulum in root geotropism[END_REF] . However recent investigations have highlighted that the gravitropism answer is independent of gravity intensity and thus that the mechanism behind gravity sensing in plants does not rely on a pressure applied by statoliths as a force sensor would do [START_REF] Chauvet | Inclination not force is sensed by plants during shoot gravitropism[END_REF] . In this framework, the hypothesis of an inclination sensor able to detect statolith position rather than pressure was recently proposed [START_REF] Pouliquen | A new scenario for gravity detection in plants: the position sensor hypothesis[END_REF] .

To date, several space experiments dealing with gravity sensing to date have focused on identifying a minimum dose (i.e. the product of gravity intensity with time) associated with gravitropic response [START_REF] Volkmann | Graviresponse of cress roots under varying gravitational forces[END_REF][START_REF] Volkmann | Gravisensitivity of cress roots[END_REF][START_REF] Driss-Ecole | Gravisensitivity and automorphogenesis of lentil seedling roots grown on board the International Space Station[END_REF][START_REF] Laurinavicius | Determination of the threshold acceleration for the gravitropic stimulation of cress roots and hypocotyls[END_REF] . These space experiments, coupled with Earth experiments using a clinostat, suggest that the gravitropic response is dependent upon the dose of gravity applied. These observations are fully compatible with a sensing based on statolith repositioning when thinking in terms of statolith avalanches [START_REF] Pouliquen | A new scenario for gravity detection in plants: the position sensor hypothesis[END_REF] . The microgravity environment indeed offers the opportunity to nullify any pressure applied by statoliths and further tests the importance of statolith pressure in triggering the gravity transduction pathway. In the case of an inclination sensor detecting statolith positioning, a mechanism of direct or close interaction between statoliths and another cell component in triggering the gravity transduction pathway is expected. Several proteins located on plastids membranes are potential candidates for statolith position detection [START_REF] Strohm | A functional TOC complex contributes to gravity signal transduction in Arabidopsis[END_REF][START_REF] Singh | Striking the right chord: signaling enigma during root gravitropism[END_REF] . Moreover, statoliths are known to be displaced toward the shootward part of the statocytes in microgravity (i.e. toward the nucleus), presumably by an actin cytoskeleton-mediated process [START_REF] Driss-Ecole | Lentil root statoliths reach a stable state in microgravity[END_REF] . Thus, microgravity can also be used for removing contacts between sedimented statoliths and any cell component without any change in orientation.

Calcium is considered to be an important actor in response to environmental changes. However, the results seem to be contradictory concerning the role of Ca in the gravity signal transduction. Initially, the concentration of cytosolic free calcium, [Ca 2+ ] c, changes were initially not detected in Arabidopsis root caps [START_REF] Legué | Cytoplasmic free Ca 2+ in Arabidopsis roots changes in response to touch but not gravity[END_REF] . More recent advances in Ca 2+ imaging methods have enabled the detection of [Ca 2+ ] c in response to a gravistimulation [START_REF] Toyota | Gravitropism and mechanical signaling in plants[END_REF] , and [Ca 2+ ] c increases were later confirmed in Arabidopsis roots [START_REF] Monshausen | Dynamics of auxin-dependent Ca 2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis[END_REF] . The gravistimulation triggered by a modification of plant inclination induces two peaks of [Ca 2+ ] c changes during the minutes following gravistimulation in young Arabidopsis seedlings [START_REF] Plieth | Reorientation of seedlings in the earth's gravitational field induces cytosolic calcium transients[END_REF] . Using parabolic flights, Toyota and co-workers [START_REF] Toyota | Analyses of a gravistimulation-specific Ca 2+ signature in Arabidopsis using parabolic flights[END_REF] showed that the first transient [Ca 2+ ] c peak was caused by the change in orientation while the second sustained [Ca 2+ ] c peak was due to gravistimulation. Interestingly, these works suggest that the Ca 2+ also triggers downstream changes, reflects auxin-triggered signaling during the gravitropism response which changes the orientation of seedling. Thus, space experiments offer the advantage of inducing gravity stimulus without any change in orientation of the seedling.

In this study, Brassica napus L. seedlings were grown aboard the International Space Station (ISS) either in simulated gravity (on a centrifuge with and acceleration equivalent to 1 g) or in microgravity. Modifications of statolith positioning were achieved by (i) removing gravity in roots grown on a centrifuge by stopping the simulated gravity, (ii) subjecting roots grown in microgravity to a centrifuge acceleration. The experiment took advantage of the fixative injection being automatically accomplished in the centrifuge without human intervention, therefore avoiding artefactual statolith repositioning. A 10 minute duration time was chosen so that the end point measurements were made after the triggering of the gravity transduction pathway. Our previous experiments, dedicated to finding the presentation time (i.e. the minimum duration of gravistimulus needed to induce a gravitropic response) for gravistimulated rapeseed seedlings, indicated that a time period of one to two minutes of gravistimulation is sufficient for triggering a gravitropic response [START_REF] Legué | Modification of the gravitropic response of seedling roots of rapeseed (Brassica napus) transformed by Agrobacterium rhizogenes A4[END_REF] . In the same way, the experiments performed in parabolic flights highlighted transient intracellular calcium fluctuations within the first minute following gravistimuli [START_REF] Toyota | Analyses of a gravistimulation-specific Ca 2+ signature in Arabidopsis using parabolic flights[END_REF] .

To access the effect of the position or displacement of statoliths on the trigger of calcium-dependant pathways, the changes in the amount of cytoplasmic free calcium were monitored by using a method specific for calcium precipitation in plants namely the pyroantimonate (PA) precipitation method [START_REF] Legué | Modification of the gravitropic response of seedling roots of rapeseed (Brassica napus) transformed by Agrobacterium rhizogenes A4[END_REF] .

Results

Root growth and root apex orientation. Brassica napus seeds were germinated in culture chambers (CCs) set up in the Kubik facility (Fig. 1A,B). Forty hours after hydration in simulated gravity condition (1 g s ), the mean length of Brassica napus primary root was 5.9 ± 0.2 mm (mean ± se, n = 44) (Table 1). No significant difference (P = 0.085) was found in primary root lengths between microgravity (µg) and 1 g s . Root lengths were also statistically similar between the 10 minute of simulated gravity onset and removal (µg + 1 g s and 1 g s + µg) and their respective control (1 g s and µg).

Figure 1D illustrates the angle distribution relative to the simulated gravity vector of all root apices at the end of the experiment. Roots growing in 1 g s conditions were all directed toward the simulated gravity vector, while apices of roots growing in microgravity conditions spread over all directions (Fig. 1D). The observed distribution of root apex orientation in µg + 1 g s and in 1 g s + µg, were close to their respective control and with similar standard deviation (Supplementary Fig. S1). Thus, a change in the root apex orientation was not detected after the 10 minutes of simulated gravity onset and removal, suggesting that only early signalling events occurred within this period.

Potassium pyroantimonate precipitates in root statocytes.

To monitor the changes in the content of cytoplasmic free calcium, we used a method specific for calcium precipitation in plants namely the pyroantimonate (PA) precipitation method [START_REF] Legué | Modification of the gravitropic response of seedling roots of rapeseed (Brassica napus) transformed by Agrobacterium rhizogenes A4[END_REF][START_REF] Wick | Selective localization of intracellular Ca 2+ with potassium antimonate[END_REF] . PA precipitates have the advantage of being electron-dense structures with a round shape, facilitating their observation and counting on ultrathin sections using transmission electron microscopy (Fig. 2) and their detection from image analysis. The presence of calcium ions in PA precipitates was confirmed by X-ray microanalysis (Supplementary Fig. S2).

The observation of electron microscopy images revealed that PA precipitates were mainly distributed in the cytosol of root statocytes (Fig. 2A,E). Some precipitates were also observed inside cell organelles as nuclei (Fig. 2A,F) and vacuoles (Fig. 2D). No precipitates were observed in statoliths (Fig. 2C,G) or in the cell wall (Fig. 2B). This intracellular distribution was observed in all conditions including the simulated gravity onset and removal conditions. The low number of precipitates in each organelle did not allow for the statistical evaluation of the changes in PA precipitate repartition.

The counting of PA precipitates using image analysis (Table 2) showed that their density was similar in statocytes of roots grown for 40 h in microgravity conditions (µg) and under simulated gravity 1 g s (P = 0.812). On the other hand, a significant four-fold increase was observed in the 10 minutes simulated gravity condition (µg + 1 g s , P = 0.024) compared to the µg condition. A significant two-fold increase was also observed in the 10 minute microgravity condition (1 g s + µg, P = 0.019) compared to the 1g s condition. These increases clearly show that Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet Statolith positioning in root statocytes. Our space experiment allowed us to acquire and analyse the coordinates of a total of 664 statoliths (Supplementary Fig. S3). No change in root orientation was reported after the 10 minutes of gravistimulation onset and removal (Fig. 1D). We therefore considered the final root orientation (and thus of statocytes) to be representative of the root orientation before the 10 minutes of simulated gravity onset and removal. We then transformed each statolith position from the image coordinate system to the statocyte coordinate one in which the origin is located at the centroid of the cell, and the y-axis is aligned with the longitudinal direction of the cell (i.e. the longitudinal axis of the root). For each experimental condition, the position of all statoliths in the statocyte coordinate system was represented (Fig. 3A-D; Supplementary Fig. S3). µg + 1 g s 5.5 a ± 0.4 9

Table 1. Root length of Brassica napus seedlings at the end of the experiment. B. napus seedlings were grown for 40 h in simulated gravity (1 g s ); 39 h 50 min in simulated gravity followed by 10 minutes in microgravity (1g s + µg); 40 h in microgravity (µg); 39 h 50 min in microgravity followed by 10 minutes in simulated gravity (µg + 1 g s ). Data presented are mean ± se. By column, means followed by a same letter are not significantly different at a 5% level using a Student-Newmans Keuls test. Roots that were partially hidden by foreground elements in the pictures were not measured.

Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet-Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity. Scientific Reports, 8, 10. , DOI : 10.1038/s41598-018-29788-7

As expected in Brassica napus roots growing in simulated gravity (1 g s ), a polarized structure of statocytes with a nucleus in the shootward side of the cell was observed. A voluminous vacuole occupied the cell (Fig. 2). All statoliths were localized in the rootward half of each statocyte (no statolith was positioned above zero along the cell longitudinal direction; Fig. 3A). Statoliths were equally distributed along the transverse section (55% in left side, 45% in right side), resulting in all roots being in the same direction than the simulated gravity vector (Fig. 1D). Surprisingly, most of the statoliths of roots growing in microgravity (µg) were also located in the rootward part of each statocyte, with only 18% of statoliths above the position of the cell centroid (Fig. 3B). However, the longitudinal distribution of statoliths in microgravity showed a long distribution tail for higher values, while in simulated gravity the distribution along the longitudinal cell direction was visually close to a Gaussian distribution (count histograms in Fig. 3A,B). In addition, the mean relative position of statoliths along the longitudinal direction from roots grown in microgravity (c.i. 95%: -4.4 ≤ µ ≤ -2.9) was significantly different from the mean position of statoliths from roots grown in simulated gravity (c.i. 95%: -7.2 ≤ µ ≤ -6.4; p-value < 0.001). Thus, the mean statolith position in Brassica napus roots growing in microgravity was from 2.0 to 4.3 µm closer to the statocyte centroid compared to roots growing in simulated gravity. Moreover, the standard deviation was a useful indicator to show that in the population of statoliths in simulated gravity, there was less of a spread than in the population of statoliths in microgravity (s.d. = 3.1 µm in 1 g s and 4.6 µm in µg).

Statolith positioning in roots that were subjected to 10 minutes of simulated gravity onset and removal (µg + 1 g s or 1 g s + µg) were compared to their respective initial conditions (µg or 1 g s ) (Fig. 3). No statistical difference was found in the mean positioning of statoliths: neither in the longitudinal, nor in the transverse direction in cells of roots submitted to 10 minutes of microgravity (1 g s + µg), compared to their controls (1 g s ). Similarly, statolith positioning after 10 minutes of simulated gravity (µg + 1 g s ) were not statistically different from that observed in microgravity (µg, Fig. 3C). Given the higher variances and the size of samples, the minimum displacement that would have been detected is 1.0 µm after 10 minutes of microgravity and 1.8 µm after 10 minutes of simulated gravity in this experimental design. These results suggest that both simulated gravity onset and removal induced a statolith displacement of low amplitude. Statoliths coordinates were also transformed along the centrifugation force (centripetal coordinates) but still no significant displacement toward the centrifugation force direction was observed (Fig. 3D). The dissymmetry along the transverse direction of statolith distribution in µg + 1 g s results from the higher number of roots oriented rightward in this condition (39% on the left side, 61% on the right side; Fig. 3D count histogram). In microgravity, statoliths were equally distributed along the transverse section (51% on the left side, 49% on the right side).

In conclusion, the statolith positioning along the root longitudinal axis was statistically different between microgravity and simulated gravity statocytes. No difference in statolith positioning was found after the 10 minutes of simulated gravity onset and removal, suggesting that any expected displacement was of low amplitude or that not all statoliths were displaced.

Discussion

Root statocytes are polarized cells where the endoplasmic reticulum is located near the cell wall, mainly towards the root apex, and the nucleus is maintained at the shootward end by the cytoskeleton [START_REF] Driss-Ecole | A polarized cell: the root statocyte[END_REF] . Inclination and centrifuge experiments undoubtedly showed that statoliths settle in the direction of the gravity vector independently of the root statocyte polarity. In microgravity, though, the positioning of statoliths reaches a stable state close to the nucleus in lentil roots [START_REF] Driss-Ecole | Lentil root statoliths reach a stable state in microgravity[END_REF][START_REF] Lorenzi | Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity[END_REF][START_REF] Perbal | Graviperception of lentil seedling roots grown in space (Spacelab Dl Mission)[END_REF] . Statolith grouping near the cell center in microgravity was also confirmed from 3D reconstruction of root statocytes of white clover [START_REF] Smith | Modulation of statolith mass and grouping in white clover (Trifolium repens) grown in 1-g, microgravity and on the clinostat[END_REF] . Consistent results were found in the current study where statoliths were more spread out in microgravity and their mean position was significantly closer to the cell center. However, it can be noticed that statolith positioning in microgravity was surprisingly close to statolith positioning under simulated 1 g s with only 18% of statoliths towards the nucleus. One hypothesis is that the intracellular space available for statolith displacement was limited by the surrounding organelles and, notably, by the presence of vacuoles that occupied most of the intracellular space. The presence of a large central vacuole in stem statocytes and its close interaction to statolith dynamics is known as stem statocyte [START_REF] Saito | Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem[END_REF] . In root statocytes, vacuoles are expected to be smaller and less present than in surrounding cells, including meristematic cell, cells of the lateral columella and cells of the outer root cap [START_REF] Sack | Plant gravity sensing[END_REF][START_REF] Griffiths | Organelle distribution in the statocyte cells of the root-tip of Vicia faba in relation to geotropic stimulation[END_REF] . Large vacuoles in root statocytes may be either a characteristic of rapeseed or a consequence of cultivation in microgravity. This observation has already been noted in microgravity with root statocytes from soybean seedlings [START_REF] Klymchuk | Changes in vacuolation in the root apex cells of soybean seedlings in microgravity[END_REF] . Another hypothesis is that the motor of statolith displacement towards the nucleus in microgravity is weaker in rapeseed as already reported in lentil seedlings [START_REF] Driss-Ecole | Lentil root statoliths reach a stable state in microgravity[END_REF] .

Our analysis also highlights that neither the onset of simulated gravity after microgravity (µg + 1 g s ) nor the removal of simulated gravity for 1 g s roots (1 g s + µg) induced a detectable or significant statolith displacement compared to the initial position of statolith. In fact, no displacement or rather low displacement was observed. Statolith velocity reported after changes of inclination is in the range of 0.3-1.2 µm min -1 for Lepidium [START_REF] Iversen | Movement of amyloplasts in the root cap cells of geotropically sensitive roots[END_REF] . In maize, it decreases from 19.1 µm min -1 to 3.7 µm min -1 , a few seconds to 30 seconds after change of inclination [START_REF] Sack | Amyloplast sedimentation kinetics in gravistimulated maize roots[END_REF] . Statolith sedimentation has been reported to be over after 400 seconds in Arabidopsis roots [START_REF] Zheng | Dynamical and microrheological analysis of amyloplasts in the plant root gravity-sensing cells[END_REF] . Accordingly, the mean difference of 3.2 µm observed between statolith positioning in microgravity and simulated gravity should disappear when microgravity roots are submitted to ten minutes of simulated gravity. However, one should keep in mind the variability of root apex orientation in microgravity relative to the centrifuge rotor, which prevents the use of the microgravity condition as a consistent starting point. When root apex orientation relative to the centrifuge rotor is homogeneous (1 g s and 1 g s + µg gravity condition), it is easier to highlight a displacement of statoliths. Statolith velocity from stimulated gravity to microgravity was previously estimated at close to 0.15 µm min -1 by Perbal and Driss-Ecole in lentil seedlings [START_REF] Driss-Ecole | Lentil root statoliths reach a stable state in microgravity[END_REF] . According to this range of velocity, statolith displacement after 10 minutes of microgravity is sufficient to remove contacts with another cell component, but still too low for being detected in this study. All these observations sustain that in rapeseed a very low displacement of statoliths occurred or that only a small number of statoliths were displaced. The latter is in accordance with the concept of "lead statoliths" where statoliths at the forefront are more prone to be displaced during the first minutes following a change of inclination [START_REF] Volkmann | Statoliths motions in gravity-perceiving plant cells: does actomyosin counteract gravity?[END_REF] . According to previous experiments done in microgravity, the combination of threshold values for gravity sensing with statolith position in microgravity sustains that low displacement of statoliths of similar amplitude (below 1 µm) is sufficient to trigger the downstream transduction pathway of gravity sensing [START_REF] Hilaire | Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D[END_REF] .

The pyroantimonate (PA) method was successfully used to study cellular and subcellular localization of calcium in gravistimulated organs. Previous comparisons between microgravity and 1 g s grown roots highlighted a decrease of the density of PA precipitates in microgravity, especially in the nucleus and cytosol while the density of PA precipitates increases in the vacuole [START_REF] Lorenzi | Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity[END_REF][START_REF] Slocum | Cellular and subcellular localization of calcium in gravistimulated oat coleoptiles and its possible significance in the establishment of tropic curvature[END_REF] . Similar observations were made in response to simulated microgravity using clinostat [START_REF] Perbal | Graviperception of lentil seedling roots grown in space (Spacelab Dl Mission)[END_REF] . Experiments including short gravistimuli in oat coleoptiles grown on Earth showed PA precipitate relocalization within 30 minutes in the bending region after a change of inclination [START_REF] Slocum | Cellular and subcellular localization of calcium in gravistimulated oat coleoptiles and its possible significance in the establishment of tropic curvature[END_REF] . Here, the density of PA precipitates was similar in both the simulated gravity and the microgravity conditions, representing conditions with stable levels of gravity during the whole experiment. Following the gravistimulus induced by the centrifuge, we observed a significant four-fold increase of the PA precipitate density, whereas a very slight statolith displacement was observed. These observations hypothesize that the establishment of contacts between the statoliths and any close cell component may be of importance for triggering the release of calcium. PA precipitate density also increased by a two-fold factor after removal of the simulated gravity i.e. after removal of contacts and nullifying the weight of statoliths. This observation indicates that the gravity transduction pathway might be linked to statolith motion and reposition while discarding mechanisms based on the detection of a force applied by statoliths, confirming previous works [START_REF] Chauvet | Inclination not force is sensed by plants during shoot gravitropism[END_REF] .

In conclusion, this study suggests that statolith displacements of very low amplitude were sufficient to trigger the release of intracellular free calcium after simulated gravity onset. This release of intracellular free calcium is expected to be the cause or/and the consequence of the triggering of the gravity transduction pathway.

Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet-Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity. Scientific Reports, 8, 10. , DOI : 10.1038/s41598-018-29788-7

Interestingly, this result was also observed after simulated gravity removal, indicating that the weight of statoliths may not be essential for gravity sensing. This observation provides valuable information about the variable that could be sensed by the statocytes. A first hypothesis could be the sensing of statolith repositioning through contact establishment or removal with another cell component. A second hypothesis is the sensing of statolith motion using an intracellular network like the actin cytoskeleton. In vivo experiments coupling statolith tracking with an indicator of the gravity transduction pathway such as calcium and pH monitoring should help in disentangling the gravity sensor functioning. Identifying the reservoir of intracellular calcium that is mobilized in the gravity signal transduction is also a primary objective for upcoming studies.

Methods

Kubik facility and PolCa hardware. The space experiment, called PolCa, was performed on the Kubik facility, located in the International Space Station (ISS). Kubik is designed for automatic microgravity experiments and was built by the COMAT company (Toulouse, France) for the European Space Agency. The Kubik facility consists of a small controlled-temperature incubator with a centrifuge, which enables simultaneous 1 g control samples to run with microgravity samples (Fig. 1A). When the centrifuge was activated, a simulated gravity acceleration (g s ) was generated perpendicular to the main axis of the culture chamber (Fig. 1B,C) with an intensity equal to the Earth gravity (g) (1 g s = 1 g) at the position of the seeds. Specific experimental units (EUs,) can be inserted and removed on the centrifuge or in the incubator. Eight specific experimental units (EUs) designed and built by the COMAT Company, according to our specifications, were used for the PolCa experiment. Each EU includes a culture chamber (CC) dedicated to seedling hydration, connected to a pouch of water for the germination activation and to one or two fixative pouches (Fig. 1B). The water and fixative solutions were automatically injected in the CCs at the appropriate time according to the predetermined timeline (Fig. 4). Each CC contains a plastic seed support able to receive 7 Brassica napus seeds and it is then sealed with a transparent plastic cover.

Pre-flight ground preparation. After cleaning the CC, an appropriate piece of Whatman paper was placed in the plastic seed support of the CC. A double-sided adhesive tape was added onto the upper side of the paper in order to maintain seeds and avoid any physical displacement due to centrifuge acceleration and deceleration. Seven dry seeds of Brassica napus var Pau (Biogemma, Toulouse, France) were layered out and oriented in such a way that the embryonic root was perpendicular to the longitudinal axis of the CC (Fig. 1B). Seeds were then covered by a piece of cellulose sponge in order to facilitate germination after hydration. The prepared supports with seeds were stored in the darkness at 22 °C during 8 days.

The 2 fixatives used for the experiment were prepared in Toulouse (France). The fixative dedicated to the analysis of statolith positioning contained 3% paraformaldehyde (v/v) (Sigma, ref. P6148) and 0.5% glutaraldehyde (EMS, ref. 16210) in 0.1 M sodium phosphate buffer (Acros Organics, pH 7.2). The fixative dedicated to the precipitation of free calcium contained 3% potassium pyroantimonate (v/v) (EMS, ref. 20220), 0.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). The fixatives were stored for 8 days at 4 °C during their transport. The fixative pouches were then filled at Baikonur (Russia) with each fixative and, one day before the launch, the support with seeds was inserted in the CC. All materials were then stored at 22 °C. The water injection, leading to seed hydration, was considered as the starting point of the experiment. 39 h50 after hydration in simulated gravity, two EUs were transferred to microgravity conditions by the cosmonaut and correspond to 1 g s + µg samples. At the same time, the two EUs initially in microgravity were placed on the centrifuge, corresponding to µg + 1 g s samples. The time period of gravistimulation onset and removal corresponds to 10 minutes for each CC. Two EUs were left on the centrifuge, corresponding to control simulated gravity (1 g s ) and two others were kept in microgravity conditions, corresponding to control microgravity (µg). In each EU, fixative solutions were automatically injected from the paunch connected to the CC, leading to the fixation of all seedlings in their growth conditions at 40 h after hydration. EUs were left on the centrifuge or in microgravity for 2 h and then stored at 6 °C until being opened on Earth. The solid lines indicate the time period in simulated gravity. The dotted lines indicate the time period in microgravity.

Experimental flight timeline. The

Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet-Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity. Scientific Reports, 8, 10. , DOI : 10.1038/s41598-018-29788-7

Upon their insertion in the Kubik facility, the 8 CCs were stored an ambient temperature until the start of experiment (Fig. 4). The experiment started when cosmonaut Yuri Lonchakov inserted the EUs in the Kubik facility, triggering automatic water hydration in all EUs at the same time. Four EUs were placed on the Kubik-centrifuge dedicated for simulated gravity control (1 g s ) and 4 EUs were placed within the same Kubik incubator, in order to be exposed to microgravity conditions (µg). After 39 h50 of growth, the cosmonaut stopped the centrifuge and swapped the position of 4 EUs: two EUs initially placed on the centrifuge were placed in microgravity (1g s + µg condition). Simultaneously, 2 EUs initially in microgravity were placed on the centrifuge (µg + 1 g s condition). Forty hours after hydration, including or not the 10 minutes of gravistimulation onset and removal, fixatives were automatically injected without the interruption of experimentally controlled gravity condition. All experimental steps were performed around 26 °C (Supplementary Fig. S5). After 2 hours of chemical fixation, the 8 EUs were retrieved from the Kubik facility and stored at 6 °C until their opening at the laboratory (France).

Root apex embedding and sectioning. Upon reception, EUs were carefully opened and inspected to check fixation status. Pictures of all CCs were taken and seedling roots were collected. The first 5 mm root apices were sampled and individually immersed in culture multiwell plates containing fresh fixative for 30 minutes. Root apices were then rinsed with 0.1 M sodium phosphate buffer (pH 7.2) under stirring (3 × 15 min) and progressively dehydrated using successive immersions in graded series of ethanol (30 min each on ice) at 25% [vol/ vol], 50%, 60%, 80%, 95%, 100%. Each root apex was infiltrated progressively in LR White resin (Sigma-Aldrich, 94188-59-7) through successive incubations in graded series of LR White at 30%[vol/vol], 50% and 75% and finally in pure LR White for 12 hours per step. Samples were then embedded in 100% LR White using flat embedding capsules (VWR, 100488-336) ensuring proper apex orientation and finally hardened at 60 °C for 48 h.

For the analysis of statolith positioning, semi-thin longitudinal sections of the root cap (0.5 µm of thickness) were realized using a microtome. The sections were then stained with toluidine blue 0.1% (in sodium carbonate 2.5%, pH 10.5), and were observed under an optical microscope (Zeiss, Germany). For the analysis of calcium precipitation, ultrathin longitudinal sections of the root cap (around 100 nm of thickness) were realized using an ultramicrotome, dried overnight, post-stained with uranyl acetate for 20 minutes and rinsed with distilled water. All ultrathin sections were observed under a transmission electron microscope coupled to an X-ray microanalysis system (Philips CM12).

Image analysis.

After the opening of the EUs, pictures of all CCs were taken and analysed using ImageJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] (v.1.5, https://imagej.nih.gov/ij) to determine the root length and the orientation of the root apex.

The root cap of Brassica napus seedlings contains seven cell layers from the quiescent center to the root apex. Statolith positioning was analysed on the two central cells of layer 4 and layer 5 corresponding to a total of 4 cells; Supplementary Fig. S6). The location of these four cells in the central columella make them good candidates for being statocytes [START_REF] Sievers | Ultrastructure of gravity-perceiving cells in plant roots[END_REF][START_REF] Blancaflor | Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity[END_REF][START_REF] Perbal | Ultrastructure des cellules perceptrices de la gravité dans l' épicotyle d'Asparagus officinalis[END_REF][START_REF] Slocum | An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique[END_REF] . Images at high magnification (100×) were used to determine statolith positioning within the statocytes. Images were first contrasted and converted to binary images. Cell membrane and statoliths were then manually segmented to extract the coordinates of the centroids of these cellular components. As the root orientation (and by extension statocytes orientation) was not conserved during the embedding steps, images at intermediate magnification (40×) were used to extract the section orientation. Coordinates along the section orientation were rotated relative to the root longitudinal direction or relative to the centrifuge centripetal axis using the pictures of the CCs previously described.

The PA method was proved useful to evaluate the impact on the cytoplasmic free calcium content and localization in response to gravity alterations or inclination [START_REF] Dauwalder | Cellular and subcellular localization of calcium in gravistimulated corn roots[END_REF][START_REF] Klymchuk | Cytochemical localization of calcium in soybean root cap cells in microgravity[END_REF][START_REF] Hayatsu | Cytochemical and electron probe X-ray microanalysis studies on the distribution change of intracellular calcium in columella cells of soybean roots under simulated microgravity[END_REF] . The PA method is highly sensitive, allowing the detection of the precipitation of cytoplasmic free calcium at concentration down to 10 -6 M, which is above cellular concentration in the cytosol (10 -7 M range) and under cellular concentration in the nucleoplasm and vacuole 44 (10 -3 M range). During signal transduction, concentration can vary locally in the cytosol above 10 -6 M and, thus, induces formation of PA precipitates [START_REF] Klein | Critique on the K-pyroantimonate method for semiquantitative estimation of cations in conjunction with electron microscopy[END_REF] . The PA method also maintains a direct relationship between [Ca 2+ ] c and [Ca 2+ ] recovered from precipitates, linear for concentration in the mM range [START_REF] Dauwalder | Cellular and subcellular localization of calcium in gravistimulated corn roots[END_REF][START_REF] Klymchuk | Cytochemical localization of calcium in soybean root cap cells in microgravity[END_REF] . Thus, the number of PA precipitates is a good proxy of the quantity of [Ca 2+ ] c and can be used for comparisons between our different conditions.

The analysis of pyroantimonate (PA) precipitates was done on statocytes of the layer 4 of the root cap. The quantity of PA precipitates was determined in each cell from the transmission electron microscope images taken at a magnification of 3810×. To compare cells with different sizes, the density of pyroantimonate precipitates was determined for an area of 500 µm² corresponding to the average statocyte surface.

Statistical analysis.

For all traits considered, as root length, density of precipitates and statoliths coordinates, the data were analyzed by using a one-way analysis of variance (ANOVA) with the PROC GLM procedure of SAS (v9.3, SAS Institute Inc, Cary, NC, USA, 2006). The homogeneity of variances was tested using the Levene's test. When the assumption of homogeneity of variance was rejected or when unbalanced data were performed between groups, a Welch's Anova was used instead of the usual Anova to test for differences between group means. Means obtained for the different gravity conditions were separated using a Student-Newman-Keuls multiple comparisons test when a main effect was determined to be significant at a 5% level.

Estimation of the least significant displacement of statoliths that could have been detected was done by generating i theoretical samples having same size and standard deviation as experimental samples. Draws were done on a theoretical Gaussian distribution and an increasing difference in the mean of each condition was generated by step of 0.1 µm (i = 10 000 per step). Fischer t-test was used to test the difference between samplings on each draw. Least significant displacement was reported when Fischer t-test detected a difference in at least 80% of the draws (Statistical power 1 -β = 0.8 and α = 0.05).

Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet-Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity. Scientific Reports, 8, 10. , DOI : 10.1038/s41598-018-29788-7

Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet-Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity. Scientific Reports, 8, 10. , DOI : 10.1038/s41598-018-29788-7

Figure 1 .

 1 Figure 1. Kubik instrument, culture chambers and root apex orientation at the end of the experiment. (A) Inside of the Kubik facility highlighting the position of the experimental units (EUs) along the centrifuge rotor. (B) EU with a view on the culture chamber (CC) located at the top right-hand side. Water and fixatives pouches are hidden inside the EU. (C) Representative CCs after 40 h after hydration in simulated gravity (top) and microgravity (bottom) conditions. (D) Distribution of root apex orientation per class of 15° angle for each gravity condition (each tile stands for one root). The red arrow indicates the direction of rotation of the Kubik centrifuge. The blue arrows indicate the direction of the simulated gravity vector. SeeTable 1 for description of growth conditions.

Figure 2 .

 2 Figure 2. Representative transmission electron microscopy images of the root statocytes of Brassica napus seedlings with the presence of potassium pyroantimonate (PA) precipitates. Images are shown for seedlings grown in simulated gravity (A-C), microgravity (D) or after simulated gravity removal (1 g s + µg, E-G). Thanks to the electron-dense structure, PA precipitates are visible as spherical black dots and were mainly observed in the cytosol (A and E). Some precipitates were detected near statoliths, inside the vacuoles (D) and the nucleus (F). No precipitates were found either in the cell wall (B) or in the statoliths (C). Arrows indicate examples of PA precipitates in various cellular components. C: cytosol; CW: cell wall; N: nucleus; S: statoliths; V: vacuole. Bar scales indicate 2 µm.

Figure 3 .

 3 Figure 3. Statolith positioning within root statocytes. The position of each statolith is indicated in blue and is given relative to the statocyte centroid (0,0) along the cell transverse and longitudinal directions. Data are shown for each condition: 1 g s (A), µg (B), 1 g s + µg (C), µg + 1 g s (D) and are accompanied by a histogram of counts along each direction. In the µg + 1 g s (D) condition, red spots indicate the position of statoliths along the centrifugation force (centripetal coordinates). Solid lines and values indicate the means of counts for each condition.

  PolCa experiment was launched on March 26, 2009 on a Soyuz rocket on flight TMA-14, during the Expedition 19 mission and was back on earth with Soyuz capsule on the April 7, 2009. The experiment was performed on the ISS in the Kubik instrument from April 3 to April 6, 2009.

Figure 4 .

 4 Figure 4. PolCa experimental timeline as performed in the Kubik facility.The cosmonaut positioned four experimental units (EUs) on a centrifuge and four EUs in microgravity conditions, triggering simultaneously an automatic water injection in the CCs. The water injection, leading to seed hydration, was considered as the starting point of the experiment. 39 h50 after hydration in simulated gravity, two EUs were transferred to microgravity conditions by the cosmonaut and correspond to 1 g s + µg samples. At the same time, the two EUs initially in microgravity were placed on the centrifuge, corresponding to µg + 1 g s samples. The time period of gravistimulation onset and removal corresponds to 10 minutes for each CC. Two EUs were left on the centrifuge, corresponding to control simulated gravity (1 g s ) and two others were kept in microgravity conditions, corresponding to control microgravity (µg). In each EU, fixative solutions were automatically injected from the paunch connected to the CC, leading to the fixation of all seedlings in their growth conditions at 40 h after hydration. EUs were left on the centrifuge or in microgravity for 2 h and then stored at 6 °C until being opened on Earth. The solid lines indicate the time period in simulated gravity. The dotted lines indicate the time period in microgravity.

  -Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity.

	Scientific Reports, 8,
	10. , DOI : 10.1038/s41598-018-29788-7

Table 1

 1

	Condition	Root length (mm)	n
	1 g s	5.7 a ± 0.3	11
	1 g s + µg	6.7 a ± 0.5	11
	µg	5.4 a ± 0.3	13

for description of growth conditions.

Condition Density of precipitates (number per 500 µm² area)

			Number of
			analysed cells
	1 g s	9.2 a ± 1.3	14
	1 g s + µg	16.9 b ± 2.7	14
	µg	9.8 a ± 2.3	12
	µg + 1g s	44.1 b ± 11.9	8

Table 2 .

 2 Density of calcium precipitates in root statocytes, expressed as numbers of precipitates by a 500 µm 2 protoplasm unit area. B. napus seedlings were grown for 40 h in simulated gravity (1 g s ); 39 h 50 min in simulated gravity followed by 10 minutes in microgravity (1 g

s + µg); 40 h in microgravity (µg); 39 h 50 min in microgravity followed by 10 minutes in simulated gravity (µg + 1 g s ). Data are means ± se. By column, means followed by a same letter are not significantly different at a 5% level using a Student-Newmans Keuls test.

Comment citer ce document : Bizet, F., Pereda-Loth, V., Chauvet-Thiry, H., Gérard, J., Eche, B., Girousse, C., Courtade, M., Perbal, G., Legué, V. (2018). Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity. Scientific Reports, 8, 10. , DOI : 10.1038/s41598-018-29788-7

SciEntiFic REPORTs | (2018) 8:11442 | DOI:10.1038/s41598-018-29788-7

SciEntiFic REPORTs | (2018) 8:11442 | DOI:10.1038/s41598-018-29788-7both gravistimulation onset and removal triggered an increase in the number of PA precipitates, suggesting an increase in free cytosolic calcium in root statocytes.

Acknowledgements

This work is supported by the French Space Agency (CNES) and by the European Space Agency (ESA). This studies is also supported by the ANR Blanc Grap2 (ANR-13-BSV5-0005-01), the European Research Council under the European Union Horizon 2020 Research and Innovation programme (grant agreement No 647384) for HC. We would like to thank G. Gauquelin-Koch (CNES) for her support, D. Chaput (CNES), the COMAT and ESA teams and G. Gasset (GSBMS) for the technical assistance and for the preparation of the experiment. We also thank D. Driss-Ecole, E. Badel and B. Moulia for helpful discussions.

Author Contributions

F.B., V.L. and V.P.L. wrote the main manuscript text, F.B. and H.C. analyzed statolith positioning, V.P.L. analyzed root growth and potassium pyroantimonate precipitate density, V.P.L., V.L., J.G. and B.E. did the ground preparation and histological sectioning, C.G. and F.B. did the statistical analyses. F.B., H.C., G.P., M.C., V.L. and V.P.L. reviewed the manuscript.

Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29788-7.

Competing Interests:

The authors declare no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Quy Thy Truong
email: quy-thy.truong@ign.fr

Guillaume Touya
email: qguillaume.touya@ign.fr

Cyril De Runz
email: cyril.de-runz@univ-reims.fr

© Quy

Thy Truong

Towards Vandalism Detection in OpenStreetMap Through a Data Driven Approach

Keywords: 2012 ACM Subject Classification Human-centered computing → Collaborative content creation, Computing methodologies → Anomaly detection Vandalism, Volunteered Geographic Information, Outlier detection Digital Object Identifier 10.4230/LIPIcs.GIScience.2018.61

Vandalism is a phenomenon that has affected by now the digital domain, in particular in the context of Volunteered Geographic Information projects. This paper aims at proposing a methodology to detect vandalism in the OpenStreetMap project. First, an analysis of related works sheds light on the lack of consensus when it comes to defining vandalism in VGI from both conceptual and practical points of view. Second, we present experiments on the use of clustering-based outlier detection methods to identify vandalism in OSM. The outcome of this study focuses on choosing the right variables when it comes to detecting vandalism in OSM.

Introduction

Skepticism toward the use of Volunteered Geographic Information (VGI) stems from the lack of data qualification in VGI datasets despite the likelihood of poor quality contribution occurrences. In the case of the OpenStreetMap (OSM) project, allowing anyone to map also adds the risk of welcoming ill-intentioned contributors who impoverish the quality of the data through acts of vandalism. For instance, some of the Pokemon Go players who signed up as OSM contributors wrongly mapped geographic elements in order to boost the development of Pokemon nests 1 . But how to distinguish actual vandalism from unintended mistakes? And how to automatically detect real vandalism in OSM? This paper's contribution is twofold: first we highlight the various definitions of vandalism that were adopted to automatically detect vandalism. Then we investigate the ability of an unsupervised method to detect vandalism in OSM by using a clustering-based outlier detection.

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Towards Vandalism Detection in OpenStreetMap Through a Data Driven Approach

Understanding vandalism: related work

Historically, vandalism comes from Germanic barbarians, called Vandals, who were reputed for sacking artworks and monuments during their invasions in Western Europe [START_REF] Philip | A Social-Psychological analysis of vandalism: Making sense of senseless violence[END_REF]. Over times, its meaning broadened and nowadays vandalism refers generally to material defacement made by human beings. However, a degradation does not necessarily fall under vandalism because depending on the context, an act will not bear the same label. For instance, animal slaughter can be labeled as vandalism unless the killer has a license to hunt [START_REF] Philip | A Social-Psychological analysis of vandalism: Making sense of senseless violence[END_REF]. Actually, labeling an action as vandalism requires to assess the damage caused, the author's motives and the context of the incident [START_REF] Ballatore | Defacing the map: Cartographic vandalism in the digital commons[END_REF]: these notions are already difficult to evaluate juridically, as each case has its own elements of context and oftentimes the author's motives are not directly accessible. Actually, vandalism definition is quite clear but as it relies on elements that are hard to assess for human beings, detecting it automatically remains a challenge. Automatic detection of vandalism has been widely studied in Wikipedia [START_REF] Adler | Wikipedia vandalism detection: Combining natural language, metadata, and reputation features[END_REF] and Wikidata [START_REF] Heindorf | Vandalism detection in wikidata[END_REF], but as these papers dealt with vandalism detection using supervised machine learning, they did not focus on giving a clear definition of vandalism. Actually, the existence of a corpus of labeled data on Wikidata/Wikipedia enabled them to evacuate the question. [START_REF] Neis | Towards automatic vandalism detection in OpenStreetMap[END_REF] developed a rule-based vandalism detection system for OSM data. The rules mainly take into account user reputation and object history. Therefore OSM newbies' created objects are at a disadvantage as they are more prone to be detected as vandalism. Unlike Wikidata, no corpus of OSM vandalism data is available. This is why our experiment attempts to detect OSM vandalism with the use of an unsupervised method and considering other vandalism metrics that were not tackled in [START_REF] Neis | Towards automatic vandalism detection in OpenStreetMap[END_REF].

Through the analysis of intentional vandalism incidents on Wikimapia and OSM, [START_REF] Ballatore | Defacing the map: Cartographic vandalism in the digital commons[END_REF] proposed a typology for carto-vandalism composed of six categories: play carto-vandalism, ideological carto-vandalism, fantasy carto-vandalism, artistic carto-vandalism, industrial carto-vandalism and spam. This typology is drawn from experimental observations so it is quite realistic, however it can be difficult in some cases to label vandalism straight away in one of these categories. For instance, artistic carto-vandalism can be seen as a sub-category of fantasy carto-vandalism: mapping polygon art is necessarily a fictional data. In fact, the proposed typology implies knowing the contributor's intentions, which is a research problem in itself. On this intentionality issue, [START_REF] Neis | Towards automatic vandalism detection in OpenStreetMap[END_REF] solves the problem by stating that " in the case of OSM, vandalism can occur intentional and unintentional, contradicting the traditional definition of the term 'vandalism' ". However, the OSM Wiki page about vandalism2 does mention the difference between vandalism and bad editing which lays in the contributor's purpose, although both of them require data repairs. Actually, OSM vandalism and bad editing may both result in the same defacement of the dataset. This is why OSM Wiki page on vandalism does not provide a definition of what vandalism is but how it manifests in OSM dataset together with bad editing. Thus another challenge for vandalism detection in OSM is to steer clear of mistaking bad edits with true vandalism (i.e. minimizing false positives).

Like in [START_REF] Neis | Towards automatic vandalism detection in OpenStreetMap[END_REF], we analyze some cases of OSM user blocks in the light of the context, the user's motive and the caused damage in order to better understand what belongs to true vandalism and what does not. The case depicted in Figure 1 is true vandalism on versions 6 and 7, as the contributor3 completely defaced the nature of a Russian island by changing its name tags and turning it into a park. These edits are obviously made on purpose. We also note that the changes made on version 8 are not vandalism as the same user brings back the values of some previously altered tags. The edit war on an area in Latvia depicted on Figure 2 shows a disagreement about the real nature of this place. The banned user 4 is the one who added the 'leisure=park' tag to the area. However, further research shows that local people do consider this place as a park 5 . Consequently, this case is not truly vandalism but rather highlights the ambiguity of the geographic object. Lastly, adding unconventional tags6 can be seen as an abnormal contribution but in this case it is not vandalism. Some of the tag values are understandable for humans and actually add valuable information to the objects (Figure 3). These examples show that vandalism -according to a data-oriented traditional definition -is less regular than contributions being non-compliant to OSM policy. Due to the scarcity of vandalism in OSM and the difficulty to enumerate all of the possible cases, this study tackles the vandalism issue following an outlier detection approach.

Methodology

Assuming that vandalized data form outliers in a dataset, our experiment aims at finding out whether using a clustering-based outlier detection enables to identify vandalized data in an OSM dataset. As vandalism does not often occur on OSM and we do not know where it happened, we cannot choose a study area where we would be assured to find vandalism cases. Therefore, we need to purposely add vandalized data so that the outliers to be detected are known in advance.

Then, every OSM element should be described by variables that will be used as inputs for the clustering algorithm. In the first place, the experiment will be limited to the detection of vandalism on buildings. This implies retrieving OSM ways and OSM relations that contain the 'building' key tag. To best describe OSM data, several types of descriptors may be contemplated: geometric variables [START_REF] Girres | Quality assessment of the french OpenStreet-Map dataset[END_REF], topological variables [START_REF] Girres | Quality assessment of the french OpenStreet-Map dataset[END_REF], historic variables [START_REF] Neis | Towards automatic vandalism detection in OpenStreetMap[END_REF] and user variables have been used in the literature to qualify OSM and crowdsourcing data in general [START_REF] Adler | Wikipedia vandalism detection: Combining natural language, metadata, and reputation features[END_REF][START_REF] Neis | Towards automatic vandalism detection in OpenStreetMap[END_REF]. In this study, we employ fantasy and artistic vandalism to deface our dataset so at the moment only geometric variables were input into the clustering algorithm, as artistic vandalism is characterized with oddly shaped objects (Table 1). Eventually, the clustering algorithm will group similar objects according to their input attributes while setting aside buildings having particular values.

Experiment and initial results

In this study, the dataset is composed of OSM buildings that are located in Aubervilliers, a suburban town of Paris. Vandalism committed in this dataset includes (Figure 4): 17 fictional buildings of different sizes which were mapped in a blank space (the yellow polygon in Figure 4 indicates that this space is currently an area under construction) and 10 artistically shaped buildings that were mapped in the middle of a river and over the town's graveyard. The outlier detection was run using the DENCLUE clustering algorithm (Java Smile library) because it is noise-invariant and remains efficient for high dimensional datasets [START_REF] Hinneburg | DENCLUE 2.0: Fast clustering based on kernel density estimation[END_REF]. It takes a smoothing parameter σ that describes the influence of a data point in the data space, and a parameter m that corresponds to the noise threshold. The algorithm starts by building a clustering model based on the input variables, then predicts the class of each element according to the clustering model. At this point, buildings whose descriptors are totally inconsistent with the clustering model are classified as outliers. The others are classified into clusters. However, some clusters contain only one element, meaning the values of these buildings descriptors fit into the clustering model but no building was similar enough regarding its attributes' values to belong to the same cluster. Thus, in a certain way, these one-size clusters can be considered as outliers too but to a lesser degree. Table 2 summarizes the number of outliers and one-size clusters that were detected for each kind of data (vandalism or not).

The first 'e' letter-shaped building was the only outlier-labeled vandalism while the remainder of artistically vandalized buildings -including the other two 'e' letter-shaped ones -was classified into one-size clusters. We note that 25 cases of vandalism out of 27that represents 92% of known vandalism -could be retrieved either in the outlier class or a one-size cluster, which is quite outstanding. Nevertheless, 60% of normal buildings have been also classified into outliers or one-element clusters. By taking a look at the variables of the vandalized buildings, we notice that the geometric descriptors do not bring out the geometric peculiarities of the artistic vandalism that was committed into our dataset. Maybe considering a polygon density variable which accounts for a polygon's number of vertices G I S c i e n c e 2 0 1 8

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Towards Vandalism Detection in OpenStreetMap Through a Data Driven Approach would have brought out all of the committed artistic vandalism. OSM French buildings have been mostly imported through mass imports from the French cadaster, so a lot of OSM building elements actually map small and weirdly shaped pieces of building. This is why the tiniest fictional building was not seen as an outlier given the strong presence of small sized elements in the dataset. Therefore we should reconsider geometric attributes that would not bring out the geometric specificity of geographic objects. Eventually, our input variables did not take into account the building's spatial relations with other elements. Here, some vandalized buildings are contained inside a river, a construction area and intersect a cemetery. Considering additional topological variables that express these peculiar situations might improve the detection of uncommonly located vandalized building. Actually, we did not expect to successfully detect all our vandalism cases -without any false positive -by simply using a clustering method on geometric features, so this first result is fairly encouraging.

Conclusion and future work

Our work focused on the definition of vandalism and the aspects that challenge its automated detection, such as the contributor's purpose, the context and the harm done. Initial experimental results showed that detecting OSM vandalism using an unsupervised method requires a wiser choice of the attributes to be input in the clustering algorithm. These attributes cannot be simple data quality assessment features but they have to be specifically designed for vandalism detection. Future work includes exploring the influence of the σ and m parameters of DENCLUE clustering algorithm on the outlier detection predictions. Other clustering algorithms (e.g. DBScan, BIRCH) should also be tested to check if they perform better on detecting vandalism. Besides, we intend to carry out the same experiment on OSM German buildings because most of them have been mapped by hand, so unlike OSM French buildings, they should not be divided up into small pieces: maybe in this dataset our vandalism cases would be detected. We also intend to deal with other types of vandalism, for instance vandalism through tag edits or object delete. In this case, other relevant variables should be contemplated to enrich our dataset -as mentioned previously, the set of input clustering variables should be extended with topological, semantic and historical features, as well as contributor-oriented descriptors and reference data matching indicators. However we will then have to address the curse of dimensionality issue. Eventually, in the same way as with Wikipedia vandalism, supervised learning classification techniques may be contemplated to detect vandalism in OSM.

Figure 1

 1 Figure 1 Tag history of an OSM fantasy vandalism case (source: OSM Deep History application).Each column gives to the state of an OSM object's version concerning its metadata and its tag values. Key tags are on the left. The changes are coded by colors: green stands for tag addition, yellow for tag-value edit and red for tag delete.

Figure 2

 2 Figure 2 Tag history of an ambiguous area in Latvia (source: OSM Deep History application).

Figure 3

 3 Figure 3 OSM oddly-tagged contribution (source: OSM Deep History application).

Figure 4 Table 2

 42 Figure 4 Fantasy vandalism (left image) and artistic vandalism in Aubervilliers, France.

Table 1

 1 Overview of OSM building geometric variables that are used for the experiments. N.B. : MBR stands for Minimal Bounding Rectangle.

	Variable name	Description
	perimeter_out_of _max	perimeter(building) max dataset (perimeter)
	area_out_of _max	area(building) max dataset (area)
	shortest_length_out_of _perimeter	length(shortest_edge building ) perimeter(building)
	median_length_out_of _perimeter	length(median_edge building ) perimeter(building)
	elongation	width(M BR building ) length(M BR building )
	convexity	area(building) area(M BR building )
	compacity	Miller's index: 4 * π * area(building) perimeter(building) 2

https://wiki.openstreetmap.org/wiki/Vandalism

https://www.openstreetmap.org/user_blocks/1598

https://www.openstreetmap.org/user_blocks/1328

http://www.spottedbylocals.com/riga/anninmuizas-mezs/

https://www.openstreetmap.org/user_blocks/1455

-one.org/osm-discussion-comments?uid=6310073&commented