Gravitydependent differentiation and root coils ... - Wiley Online Library

Plant Biology ISSN 1435-8603

RESEARCH PAPER

Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station G. F. E. Scherer & P. Pietrzyk €lzwissenschaften, Abt. Molekulare Ertragsphysiologie, Hannover, Germany €r Zierpflanzenbau und Geho Leibniz Universit€ at Hannover, Institut fu

Keywords Asymmetrical growth; gravity-induced differentiation; microgravity; patatin-related phospholipase-A-I (pPLA-I-1); root coils. Correspondence G. F. E. Scherer, Universit€ at Hannover, Institut €lzforschung, €r Zierpflanzenbau und Geho fu Abt. Molekulare Ertragsphysiologie, Herrenh€ auser Str. 2, D-30419 Hannover, Germany. E-mail: [email protected] Editor V. Legue´ Received: 10 June 2013; Accepted: 25 September 2013 doi:10.1111/plb.12123

ABSTRACT Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity.

INTRODUCTION In most experiments with plants in space the general expectation is that plants can grow and develop more or less normally in microgravity (Halstead & Dutcher 1987; Kiss et al. 2000; Paul et al. 2012). Good examples are growth experiments showing that seed-to-seed development is possible even though full fertility is not reached (Musgrave et al. 2000; Musgrave & Kuang 2003). Many other aspects of plant growth in space have been investigated (Brown 1993; Dutcher et al. 1994; Johnsson 1997; Ferl et al. 2002; Takahashi et al. 2000; Stutte et al. 2006; Millar et al. 2011). In most experiments wild-type plants were used. Less often, wild type (WT) and mutants were compared, even though the use of mutants in ground-based research is very common. From their phenotypes, mutants show us the key regulatory genes and what exactly these genes regulate. Often a defined stress is added to the experiment, e.g. biotic stress such as a pathogen, or abiotic stresses such as nutrient deficiency, drought or unnatural light conditions, to determine whether a gene functions in a particular physiological situation. Clearly, the few available occasions to perform plant experiments in space have severely restricted the volume of research obtained. Nevertheless, the scientific potential of investigating how certain mutants react to microgravity as a form of stress and their ability for normal development seem to be

underrated. That plants do respond to microgravity through changes in developmental steps (with lack of stimulus) has been reported in several instances, and the term ‘automorphosis’ or ‘autonomous’ development has been used for such a phenomenon (Volkmann et al. 1986; Stankovic et al. 2001; Hoson & Soga 2003; Miyamoto et al. 2005). An experiment in the International Space Station (ISS) with Arabidopsis thaliana exploited a knock-down mutant of phospholipase-A-I (ppla-I-1), a member of the patatin-related phosphosplipase A gene family (Scherer et al. 2010), which showed only a few phenotypic properties that were obviously aberrant from those of the WT on the ground, in laboratory experiments. The corresponding A. thaliana Wassilewskija (Ws) WT is known to exhibit a ‘waving’ pattern of root growth on a 45° tilted agar surface; the root tip turning from left to right in such conditions, leaving a sinusoidal wave-like pattern. One obvious property of the mutant was the formation of more root coils (full circles) on a 45° tilted agar surface in 1-g in ppla-I-1 seedlings than in WT seedlings. Although root growth patterns are easily documented as flat (two-dimensional) pictures, with the roots adhering to the agar surface, they originate from circumnutation, gravisensing and negative thigmotropism, which all contribute to waving (Migliaccio & Piconese 2001; Migliaccio et al. 2009, 2013). If the root tip, forming the next arc, continues curving, it will complete a full

Plant Biology 16 (Suppl. 1) (2014) 97–106 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands

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circle, i.e. coil. Several types of mutant are known to show increased formation of root coils, e.g. those that have gravitysensing and auxin-sensing mutations or mutations in tubulinassociated proteins (Migliaccio & Piconese 2001; Sedbrook & Kaloriti 2008; Migliaccio et al. 2013). Microtubules function in microfibril and cell wall deposition, which can exert an influence on directed growth of the root tip (Lloyd & Chan 2002). We verified that the ppla-I-1 mutant is agravitropic in the root and hypocotyl and has aphototropic hypocotyls rather than being a mutant of the group of microtubule-associated proteins. These initial observations provided the basis to propose and conduct an experiment on coil formation in a 1-g and a microgravity environment in the BIOLAB facility with the ppla-I-1 mutants. However, investigation of development in microgravity and in an identical 1-g reference centrifuge sideby-side over a relatively long time span produced unexpected results. Lateral root formation, leaf expansion and hypocotyl elongation were influenced by the absence of gravity in conjunction with relatively low light conditions in the equipment. MATERIAL AND METHODS Identification of homozygous knockout line Homozygous ppla-I-1 knockout plants in the Wassilewskija (Ws) background were isolated as described (Krysan et al. 1996). The primer sequences for isolation were: AtPLAI forward 5′-GTC GAT GTC TTC TAC ATG TTC TTC TCC AT-3′, AtPLAI reverse 5′-TTT AAC AGT CTC TCA AAC TCG TTT GCA CT-3, and the primer JL202 5′-CAT TTT ATA ATA ACG CTG CGG ACA TCT AC-3′ located in the T-DNA sequence. Yang et al. (2007) isolated two knockout alleles in the Ws background from the same collection (Krysan et al. 1996). Their T-DNA insertion mutant is highly likely to be mutant ppla-I-1, we therefore also named it ppla-I-1. For RT-PCR, total RNA from seedlings was prepared using TRIzol reagent according to the manufacturer′s instructions (Invitrogen, Carlsbad, CA, USA) and converted to cDNA with RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, Waltham, MA, USA). PCR conditions were 36 cycles of 94 °C for 15 s, 65 °C for 30 s and 72 °C for 120 s, and an additional cycle of 72 °C for 240 s. The lack of mRNA expression in the homozygous lines was shown with RT-PCR using for ppla I-1 the forward primer 5′ATG TCT TCT ACA TGT TCT TCT CCA T-3′ and the reverse primer 5′-TAT CAT ACT TAT AAG CTG CCT CAC C-3′. Actin genes ACT2 and ACT7 were used as standard to normalize RT-PCR amplification, with the primers: sense 5′- AGG ATA TTC AGC CAC TTG TCT GTG-3′and antisense 5′-AGA AAC ATT TCC TGT GAA CAA TCG-3′. Growth conditions and physiological experiments Arabidopsis seeds for ground experiments were stratified for 4 days and plated on half-strength MS medium (1% Bactoagar) supplemented with 1% sucrose. For root and hypocotyl growth experiments, plants were grown on upright Petri dishes for 3 days in the dark or for 9 days under long-day conditions (16 h light:8 h dark; 50 lmol m 2s 1) at 23 °C. After hypocotyls and roots were scanned, their lengths were measured using the program dhs-Bilddatenbank version 4.01 (Dietermann & Heuser Solutions, Greifenstein-Beilstein, Germany). 98

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For the gravitropism experiments, the plants were grown in darkness at 23 °C for 3 days on upright Petri dishes, then the plates were rotated 90° and plants grown for additional 24 h under the same conditions. Angles were determined with the program axiovision version 4.2 (Zeiss, G€ ottingen, Germany). For root coiling assays, seedlings were grown in the light on harder agar (2% w/w) containing half or 2/two-thirds (in EC on ISS) concentration of MS medium plus 1% sucrose (Murashige & Skoog 1962). Plates were stood upright for 3 days to orient the roots according to gravity and then tilted to 45° to induce coiling (Simmons et al. 1995a,b). For the gravitropism experiments with young shoots, plants were grown for 5 weeks on MS medium (1% Bacto-agar) supplemented with 1% sucrose at 23 °C under long-day conditions (16 h light:8 h dark) in a plastic container to obtain young inflorescence stems. Plants were photographed with a Canon EOS D30 camera, and angles of the gravitropic responses were determined as above. For the experiment on phototropism, plants were grown in the greenhouse for 6–7 weeks under short-day conditions (8 h light:16 h dark) until they developed strong inflorescence stems of approximately 15 cm in length. The first inflorescence stem was excised to induce a higher number of inflorescence stems on each plant. Plants were placed at 23 °C, exposed to lateral blue light (Phillips TLD 36W18 Blue, distance 20 cm, 20 lmol m 2s 1) and photographed without flash light at the times indicated. Bending angles were determined as above. For the experiment in space (timeline in Figure S1) two-thirds MS (+1.33% sucrose) was used because higher osmolarity produces more coils (Buer et al. 2003). Sterile filter strips (4-mm wide) were cut so that the middle part served as a coding pattern to identify the agar boxes (polyethersulphone, Supor-800black, cat. no. S80678; Pall Corp., Port Washington, NY, USA). Filter strips can be used to identify individual agar boxes (Figure S2). Seeds were moistened for 24 h then carefully dried. These seeds were glued with 1–2 ll gum arabic (15% w/ v) to the filter strips. These strips were mounted onto a stamplike tool (Figure S3) and wrapped in sterile foil. The seeds could be stored for up to 6 months at ambient temperature, which allowed for flexible planning into the flight schedules, however, for both flights they were stored no longer than 6 weeks, including a vernalisation period at 4 °C. Ten days prior to the shuttle start, the seeds were stowed in the shuttle at ambient temperature, and 7 days prior to the start of cultivation in orbit they were stored in the refrigerator on board at 4 °C for 7 days. Then, on the ISS, the astronaut transferred the filter strip under sterile conditions in a glove box to the agar surface. The agar boxes were screwed onto their support inside the experimental containers (ECs). The mounted ECs were placed into the centrifuges, and provided with LED light (24 h) for growth support (Figures S4–S6). The WAICO1 growth experiment started on 26 February 2008 and astronaut photos were taken on 10 March. The seedlings then grew on in microgravity until 24 March, when they were retrieved on the ground. WAICO2 started on 28 April 2010 and finished on 10 May, with astronaut photos and fixation. The inbuilt video camera on the centrifuge took low-resolution pictures (720 9 540 pixels) every day of each container, these were sent telemetrically to the control station in Cologne. A final photograph of plants in each EC was made by the astronaut on day 12 using a mirror reflex camera (3040 9 2020 pixels; Figures


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S5 and S6 show the equipment). Astronaut photos could not be repeated due to astronaut working time restrictions.


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Growth and phenotype of the mutant ppla-I-1 on the ground A characterisation of the ppla-I-1 mutant on the ground was the first objective. The mutant is delayed in gravitropic response of the hypocotyl (Fig. 1A and F) and the root (Fig. 1A and E). Transcription of pPLA-I was not influenced by tilting

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Growth of the WT and mutant ppla-I-1 on the International Space Station (ISS) for the WAICO1 and WAICO2 missions was performed in ECs on two identical centrifuges so that one provided a 1-g force while the other stood still to provide microgravity (Figures S5, S6). The growth conditions were analogous to the tilting experiments for coil induction on the ground (Fig. 4): for 3 days the agar containers in all ECs in both centrifuges were in a position equivalent to upright in 1-g. This oriented the seedlings according to the gravitational vector generated in the centrifuge: hypocotyls towards the axis of rotation (‘physiological top’) and roots towards the outer wall of the centrifuge (‘physiological bottom’). Illumination was provided continuously with LEDs (Figure S4A). Then one centrifuge was stopped so that growth proceeded in microgravity for a further 9 days (timeline in Figure S1). During that time, agar containers were tilted by 45° (driven by an electrical motor) and in the same way the agar was tilted back once per day to upright position so that seedlings could be photographed with a video camera, producing still photos of seedlings in the physiological upright position. These video pictures provided a first result on growth progress (Figures S7–S9). In the WAICO1 mission, humidity control failed so that the video photos were of limited use due to droplets on the window. Daily video photos from WAICO2, with full visibility of plants, were obtained (Figure S9), but again limited resolution only allowed recognition of the main roots. A final picture (without opening the EC), done by hand by the astronaut, was an additional record of growth in the containers after 12 days in WAICO1 (Figs 5 and S10) and WAICO2 (Figs 6 and S11). So, the final photos from the astronaut at day 12 are shown in Fig. 5 and those in Figure S11 are the main documents for development in microgravity and 1-g with ECs of mutant and WT. Nevertheless, video photos from WAICO2 and astronaut photos (Figs 6 and S9) provide a good comparison and show the reproducibility of plant development in space. In the WAICO1 mission the centrifuges had to remain stopped due to non-planned fixation failure, but all seedlings continued growing for another 15 days in microgravity and light because the centrifuge had stopped. This opened a splendid opportunity to retrieve the seedlings (‘A’ ECs) grown for 3 days in 1-g and continuously in microgravity for 24 days (see timeline in Figure S1) and conduct an additional set of investigations in the ground laboratory, after transport back to Hannover for 2 days (cooled and exposed to 1-g at 5–15 °C) of the ECs (Fig. 7, see below).

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Fig. 1. Gravitropic response of dark-grown WT and ppla-I-1 seedlings (A) and growth characteristics of WT and ppla-I-1 seedlings (C–F). A: Delayed gravitropic response in hypocotyls of dark-grown ppla-I-1 seedlings as compared to the WT. B: RT-PCR of pPLA-I mRNA from hypocotyls of 3-day-old etiolated, gravistimulated seedlings. C: Growth of hypocotyls and roots of 3day-old dark-grown ppla-I-1 seedlings (black bars) and corresponding WT (Ws) seedlings (grey bars). D: Growth of hypocotyls and roots of 9-day-old light-grown ppla-I-1 seedlings (black bars) and corresponding WT (Ws) seedlings (grey bars) (SD, n = 21–42 in C and D). E: Bending angles 24 h after gravistimulation after tilting to 90° of etiolated roots of 4-day-old darkgrown seedlings (black bars: ppla-I-1; grey bars: WT. Average angles were 54.1° for ppla-I-1 and 62.2° for WT (n = 65–73; significantly different: P < 0.005). F: Bending angles 24 h after gravistimulation of etiolated hypocotyls of 4-day-old dark-grown seedlings (black bars: ppla-I-1; grey bars: WT). Average angles were 37.0° for ppla-I-1 and 64.2° for WT (n = 45–47; significantly different: P < 0.001).

(Fig. 1B). Hypocotyl and root lengths were statistically insignificantly different in the WT and mutant, both in the dark (Fig. 1C) and in white light (Fig. 1D). Gravitropic bending of


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Fig. 3. Phototropic response of young inflorescence stems to lateral blue light exposure. Diamonds: bending of inflorescence stems of WT and ppla-I1 plants (squares). SE for each time point is indicated (SE; n = 11 for ppla-I1 and WT, pooled from three independent plantings).

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Coil formation in microgravity in the mutant and WT in microgravity

Fig. 2. Gravitropic response of inflorescence stems of 5-week-old plants growing on agar under long-day conditions. Plants were tilted by 90° to start the response. A, B: WT plants and (C, D) ppla-I-1 mutants at t = 0 min (A, C) and t = 30 min (B, D). E: Diamonds: ppla I-1; squares: WT. (SE; n = 11–18 for ppla I-1 and n = 7–11 for WT, pooled from five independent plantings). * P < 0.05, ** P < 0.01, values without asterisks are not statistically significant.

young flower stalks (Fig. 2) and phototropic bending (Fig. 3) were delayed in the mutant compared to the WT. Additional impairments of this mutant in auxin and red light sensing are described in a separate publication (Y. Effendi, K. Radatz, C. Labusch, S. Rietz, R. Wimalasekera, M. Zeidler, G.F.E. Scherer, unpublished data). Root coil formation was tested on 45° tilted agar plates. The WT seedlings grew in a ‘waving’, sinusoidal pattern and coils were rare, whereas as the mutant ppla-I-1 seedlings developed about 30% more root coils than the WT (Fig. 4A and B). Root coils showed cell file rotation (CFR) in a left-handed direction, the surface of the rhizodermis cells forming a spiral on the root surface (definitions in: Migliaccio et al. 2013; Fig. 4C). In the waving part, the sensing of CFR changed with every bend. Importantly, when we tested the phytochrome mutants phyB-9 and phyA-211 on 45° agar we found that phyB seedlings formed root coils at a higher rate (about 80%) than ppla-I-1 or phyA seedlings (about 5%; Fig. 4D and E). 100

In WAICO1, coil formation could be documented after 12 day and 9 days in microgravity with photos made by the astronaut (Figs 5A–H and S10) and later on the ground after 24 days in microgravity (Fig. 7A–D). Seedlings from WAICO2 were not retrieved as fresh material, so that only the video photos made daily in orbit and the astronaut photos after 12 days were used for evaluation (Figs 6 and S11). Despite somewhat low quality of the pictures (water droplets in WAICO1 and blurred pictures from WAICO2), several observations could be made. In both WAICO1 and WAICO2 after 12 days in 1-g, the waving growth patterns obtained in the WT and mutant were indistinguishable from experiments on the ground (Figs 5E–H and S10E–H). In microgravity, only the mutant formed coils in both missions (Figs 5A–D and 6E–H). Both WT and mutant roots grew arcuated, i.e. forming a bow with few waves pointing to the physiological ‘downward’ direction and to the left. The mutant roots, on average, covered a smaller distance from the filter strip than the WT (Figs 5C, D and 6G, H), which we noticed in WAICO1 only when we photographed the plants after 27 days (Fig. 7). Since there was no gravisensing involved, this stronger bending of the mutant must have been a response to the light source, which defined the physiological ‘top’ in the agar container. In mutant seedlings this configuration leads to formation of coils in several seedlings per container but none in WT seedlings in WAICO2 (Figs 6H and S11), while in WAICO1 (Figure S10), there were several in the WT but more in ppla-I-1. Seedlings grown for a total of 27 and 24 days in microgravity could be inspected after retrieval on the ground in Hannover. The retrieved ‘A’ ECs (designations see Figure S2) were photographed and scanned with the lid opened (Fig. 7A–D) and then the agar surface was systematically photographed with a microscope to obtain pictures of all coils. This gave us the unique


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Fig. 4. Root waving in WT (A) and waving and coiling in ppla-I-1 (B) seedlings on 2% agar tilted to 45°. (C): Single coil and a short waving part of ppla-I-1 root tip. Cell file rotation is apparent in the coil and waving part. (D): Waving in phyA-211 seedlings. (E): Coiling in phyB-9 seedlings.

opportunity to study the surface of coils. From the root surface photos (Figure S12), the percentage of CFR in root coils was quantified (Fig. 7E–G). The mutant plants had developed more coils than the WT plants. To our surprise, coils in mutants were on average smaller in diameter yet exhibited less surface CFR of rhizodermis cells.

astronaut photos taken during WAICO2, however, were useful to verify the general root pattern observed in WAICO1 in microgravity (Figure S11), and showed that lateral roots were more numerous in microgravity than in 1-g in both the WT and mutant. Development of leaves and hypocotyls at 12 and 27 days

Gravity-dependent development of leaves, hypocotyls and lateral roots in WT and mutant seedlings: development after 12 days In 1-g in WAICO1 (12 days), cotyledons of the mutant were smaller (Fig. 5J) than those of the WT, and development of secondary leaves was slightly delayed (Fig. 5G and H). Surprisingly, in microgravity cotyledons in both 1-g and microgravity were of very similar size in both genotypes (Fig. 5I), indicating a gravity-induced developmental step, perhaps additionally influenced by the low light conditions in the ECs. In WAICO2 this could not be quantified from the daily video photos (Figs 6A, B and S8 & 9). On both the video photos obtained in WAICO1 (Figures S7, S8) and the astronaut photos (Fig. 5K and L) at 12 days, it was observed that ppla-I-1 plants had longer hypocotyls in microgravity than in 1-g. Quantification (Fig. 5K and L) confirmed this. Longer hypocotyls were also observed in mutant ppla-I-1 in WAICO2 even though no influence of gravity on height was observed (Fig. 6I and J). Clear differences in petiole lengths were not observed (Fig. 5M and N). As mentioned above, in the laboratory white light (50 lmol m 2s 1) we did not observe significantly longer hypocotyls or the delayed cotyledon expansion in the mutant (Figs 1 and 4B, C). Lateral root development in WAICO1 could be analysed from the photo taken after 12 days in orbit by the astronaut (Figs 5A–H and S10). More lateral roots were apparent in microgravity in both WT and mutant roots when compared to growth in 1-g, despite the poor clarity of these photos. The photos taken by the astronaut (with all their limitations) at the end of WAICO2 support this (Figure S11). The video photos captured during WAICO2 do not seem to really support this conclusion because only main roots were recognisable and no lateral roots were apparent, indicating rather poor resolution (compare Fig. 6 with Figure S11). The

Development of more leaves as well as additional root growth at 27 days, as compared to the status at 12 days, was obvious in WAICO1 (compare Figs 5 and 7). Cotyledons in the WT and ppla-I-1 were very similar in size at 27 days, confirming the analysis at 12 days for the seedlings in microgravity. The roots of the WT plants grew further away from the light source than ppla-I-1 roots (Fig. 7A–C), as confirmed through observation of video photos from WAICO2 (Figs 6G, H and S9). After retrieval, seedlings were carefully removed and scanned. Root length, lateral root number and total length of lateral roots were obtained, but differed little between the WT and ppla-I-1 (Fig. 7G and J). Seedlings grown in 1-g could not be retrieved. No consistent data for differences in root lengths or lateral root patterns of mutants and the WT grown in microgravity were found (Fig. 7H–J). DISCUSSION Coil formation in 1-g and in microgravity Gravity induced developmental changes in roots as compared to microgravity, defining gravity as a stimulus and microgravity as the lack of a stimulus. The waving pattern of WT roots in 1-g on 45° tilted agar can be considered a suppression of root coiling, which is increased in gravisensing mutants in 1-g (Migliaccio & Piconese 2001; Piconese et al. 2003; Sedbrook & Kaloriti 2008; Migliaccio et al. 2009, 2013). Since without gravity stimulus there is no gravisensing, one hypothesis was that in microgravity both the WT and mutant would show maximum coil numbers. In WT, Arabidopsis roots growing in 1-g are guided down on a vertical agar surface by gravitropism. If growing freely into air, Arabidopsis root tips move in a right-handed spiral in elliptical curves (Mullen et al. 1998), the so-called circumnutation,


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Fig. 5. Growth of seedlings in WAICO1. Astronaut photos of seedlings were taken with a digital camera outside the centrifuges, and enlarged parts are shown. (A): WT in microgravity (A3); (B) WR in microgravity (A5); (C) ppla-I1 in microgravity (A2); (D) ppla-I-1 in microgravity (A6); (E) WT in 1-g (B3); (F) WT in 1-g (B5); (G) ppla-I-1 in 1-g (B2); (H) ppla-I-1 in 1-g (B6). Width of filter strip: 4 mm. (I–N) Quantification of cotyledon areas, hypocotyl lengths and petiole lengths (n = 10–21; SE; * P < 0.01 different in comparison to WT). A2 and B5 contained ppla-I-1, A3 and A6 WT (microgravity). B2 and B3 contained ppla-I-1 and B3 and B6 WT seedlings (1-g).

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so that when the root is flattened onto an agar surface this leads to regular lateral movement of roots, forming a sinusoidal pattern that may, however, be suppressed in vertical surface position due to gravitropism (Okada & Shimura 1990; Thompson & Holbrook 2004). Coil formation in 1-g on 45° angle agar is induced by circumnutation, which in regular intervals leads the root tip to try to penetrate the surface. This penetration attempt points int the same direction as dictated by the graviresponse of the root tip. The resulting negative thigmothropism of the root tip (Okada & Shimura 1990; Massa & Gilroy 2003) in combination with gravitropism reverses the root tip down so that the waving pattern originates (Simmons et al. 1995a,b; Migliaccio & Piconese 2001; Piconese et al. 2003; Kitazawa et al. 2005; Johnsson et al. 2009). The combined growth responses of seedlings in 1-g lead to waving induced by gravitropism, circumnutation and negative thigmotropism in the ECs in WAICO1 and WAICO2 (Figs 5E–H and 6E, F). Even the less clear astronaut photos at 12 days in WAICO1 confirmed this. In 1-g negative phototropism of 102

roots seems to be of low quantitative influence, however, it may not be totally negligible in microgravity (see below). The ppla-I-1 mutant in 1-g forms a considerable number of root coils on a 45° surface (Fig. 4B). This mutant is attenuated in the root gravitropic response so that a fraction of the root tips do not bend down regularly and, thus form a coil. We could not quantify circumnutation, but the waving pattern of the mutant in 1-g did not differ from the WT so that we conclude tentatively that circumnutation in 1-g did not differ in the WT and mutant. As a conclusion, we regard the experiment in the 1-g centrifuge as a valid control and consistent with data from the ground laboratory. In microgravity there was no gravitropism; circumnutation is slowed in microgravity (Johnsson 1997; Kitazawa et al. 2005; Johnsson et al. 2009); and negative thigmotropism is considered to be absent (Migliaccio et al. 2013). Waving, however, was also found in Arabidopsis roots growing in space (Paul et al. 2012) without any reason being given. Perhaps, negative thigmotropism is possible in space through adhesion of the older root part to the


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Fig. 6. Final video photos from WAICO2 at 12 days. A–H: Designations of genotype and gravity treatment is indicated. Bottom part is identical to top (originally transmitted photos) but treated to visualise roots in false colours. Arrows point to coils. (I, J) WT is in EC A2/A3 and B2/B3, ppla-I-1 in A5/A6 and B5/B6 in 1-g. Gravity treatment as indicated. (n = 12–15; SE).

surface, with circumnutation of the tip providing the necessary force and counterforce. Due to the limits of the instrumentation in our experiment, it must be left as an open question whether negative thigmotropism exists in microgravity. In microgravity, lack of gravisensing in both the WT and mutant should make them more similar to each other than in 1-g, where the difference in gravisensing is small but apparent (Fig. 1). However, after 12 days the mutant had more coils than the WT, which had none (Figs 5 and 6), indicating yet another difference. However, after 27 days the number of coils in the WT and mutant was similar (Fig. 7E), indicating a timedependent developmental step, with increasing coil formation after longer time in microgravity. The differences still apparent could be explained in relation to the new fact that phyB seedlings develop high numbers of coils in 1-g (Fig. 4E). Phytochrome B-dependent responses, especially the response

to shade, are compromised in ppla-I-1 (Y. Effendi, K. Radatz, C. Labusch, S. Rietz, R. Wimalasekera, M. Zeidler, G.F.E. Scherer, unpublished data). Thus it is conceivable that the mild coiling phenotype of ppla-I-1 in 1-g is related to compromised phyB-dependent responses. Moreover, auxin transport in roots is regulated by red light. In tomato seedling roots phyB2 stimulates auxin transport (Liu et al. 2011) so that phyB-compromised signalling may have a destabilizing influence on auxin concentrations leading to coil formation. A further lightdependent difference was apparent in that ppla-I-1 growth was stronger arcuated, i.e. seedlings did not grow fully ‘down’ and away from the light source in WAICO1 (Figs 5 and 7), which was also observed in WAICO2 (Fig. 6). An explanation could be provided from positive red light-induced or negative blue light-induced negative root phototropism, which has been demonstrated in space (Millar et al. 2011; Kiss et al. 2012). However, compromised phyB signalling should attenuate positive root phototropism opposite to what we have observed. Asymmetric root growth as an outcome of automorphosis Nevertheless, observing arcuated growth in microgravity in the WT and mutant as such is not surprising because Arabidopsis Ws WT roots also grow arcuated in a three-dimensional clinostat and auxin-related mutants also develop coils after extended time in a 3-D clinostat (Migliaccio et al. 2013). So, Arabidopsis roots have an intrinsic tendency to elongate asymmetrically, as suggested in the model of Lloyd & Chan (2002), because cellulose microfibrils already form a spiral in every elongated cell. Whether this is sufficient to explain coil formation and how exactly phyB-dependent signalling could influence coiling remains an open question. Both arcuated growth and coil formation must be considered as automorphosis inasmuch as they appear to be microgravity-induced (Volkmann et al. 1986; Stankovic et al. 2001; Hoson & Soga 2003; Hoshino et al. 2004; Miyamoto et al. 2005). Several authors coined the term ‘autotropism’ for tropisms in microgravity (Miyamoto et al. 2005), and coil formation can also be viewed as autotropism. The basic suggestion of the term ‘automorphosis’ is that development free of gravity stimuli is different from that in 1-g, however, this should be questioned because the plants have had no opportunity to evolve gravity-free, and it must be asked whether microgravity is rather a stress stimulus for a plant and 1-g is a ‘stimulus-free’ situation. The low numeric value of microgravity may be deceptive and not suitable to define it as ‘stimulus-free’. The CFR of rhizodermis cells near the root tip could be a potential source for a force leading to coiling or asymmetrical growth of the root tip (Fig. 1C). We observed a lack of correlation of CFR and coil formation in microgravity (Fig. 7), which thus does not support the concept that rhizodermis cells exert a force in bending the root tip. Rather, there is an intrinsic tendency for asymmetric growth in the root, as also suggested from 3-D clinostat experiments (Piconese et al. 2003; Fortunati et al. 2008; Migliaccio et al. 2009, 2013). Vegetative microgravity-induced differentiation as a form of automorphosis Some phyB functions are impaired in the ppla-I-1 mutant (Y. Effendi, K. Radatz, C. Labusch, S. Rietz, R. Wimalasekera, M. Zeidler, G.F.E. Scherer, unpublished data). This could in part


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explain the observations on development in 1-g versus microgravity in the WT and ppla-I-1. The delayed or decreased cotyledon expansion as part of a phyB-like phenotype (Neff & Chory 1998) in 1-g was abolished in microgravity (Fig. 5). Conversely, the phyB-like property of long hypocotyls in low light conditions (Neff & Chory 1998) was enhanced in microgravity. Our research on the ground showed that ppla-I-1 mutants exhibited a hypersensitive elongation in white light with an added low ratio of red:far-red (‘physiological shade’), which was absent in high ratio red:far-red light (Y. Effendi, K. Radatz, C. Labusch, S. Rietz, R. Wimalasekera, M. Zeidler, G.F.E. Scherer, unpublished data). The spectrum of the LEDs that illuminated the plants in orbit is not FR-enriched (Figure S3), however, the relative enhancement of blue and red in the WAICO1 LED spectrum and the low light conditions in the ECs (evidenced by the elongated hypocotyls) may have been conducive to shade-avoidance symptoms in this mutant prone to hypersensitive elongation in response to physiological shade. Longer hypocotyls of plants grown in microgravity were also reported in Matia et al. (2010), and explained as a higher cell number in microgravity. In a comparison of Arabidopsis Was104

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Fig. 7. A–D: Scans of growth containers after 27 days (3 days in 1-g + 24 days in microgravity) in space. E–H: Quantification of CFR on surface of coils of plants retrieved from the ISS (A2/A5: ppla-I-1; A3/A6: WT). Plants grew for 3 days at 1-g in both centrifuges, then one centrifuge stopped turning (containing the ‘A’ ECs) for 9 days, one produced 1-g further on (containing the ‘B’ ECs: not shown and not retrieved). After further 15 days of plants in microgravity in the ‘A’, ECs and were returned to the ground. After transfer by air to Hannover, plants were investigated with microscopy. Definition and quantification of CFR is shown in Figure S12. n = 6–12; SE’ * P < 0.01, different in comparison to WT.

silewskija (Ws) and Columbia (Col) WT seedlings grown in microgravity, both had an etiolated or shade-experiencing appearance (long hypocotyls and petioles) and the Ws WT had clearly longer hypocotyls and a smaller petiole angle than Col seedlings, indicating that the Ws WT was more insensitive to growth inhibition in low light in microgravity (Paul et al. 2012). In conclusion, the shade response seems to be modified by microgravity, especially in ppla-I-1. The shade symptom of long hypocotyls was more clearly developed in WAICO1 than in WAICO2 in ppla-I-1 (Figs 5K and 6I). The difference between the two was higher humidity in WAICO1, in which water droplets obscured the windows, which could have caused lower light conditions in WAICO1, thus enhancing shade symptoms. This difference probably also caused the nearly identical cotyledon areas in microgravity in both genotypes, but small cotyledons in ppla-I-1 in 1-g in WAICO1 (Fig. 5). Increased root branching and lack of a clear main root in microgravity could clearly be observed in WAICO1 (Fig. 5) and were confirmed in WAICO2 (Figure S11) when seedlings were compared to growth in 1-g had clear main roots. This was not genotype-dependent, both the WT and ppla-I-1 showed


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this response to microgravity. More lateral roots (Kiss et al. 2012) and higher root mass in microgravity have been observed previously (Levine & Krikorian 1996; Millar et al. 2011). We interpret root development as strongly influenced by auxin transport, which, in turn, is influenced by microgravity (Ueda et al. 1999; Hoshino et al. 2004) and less by light, so that changes in root development in microgravity were found in both flights and genotypes. However, lower light conditions might have influenced root coil formation after 9 days in microgravity more often in WAICO1 than in WAICO2, because the absence of phyB stimulation, as seen in a phyB mutant, causes coil formation (Fig. 4E). It remains remarkable that we, unexpectedly, found gravityinduced differences in plant differentiation, e.g. the hypocotyl length, cotyledon area, development of lateral roots and coils (Takahashi et al. 2000; Musgrave & Kuang 2003; Stutte et al. 2006; Millar et al. 2011). Is there a unifying concept that could explain the observations on gravity-dependent differentiations? We think polar auxin transport could be involved in all the responses to microgravity that we observed. Polar auxin transport is gravity dependent (Ueda et al. 1999; Hoshino et al. 2004) and is also involved in the shade-induced growth response (Keuskamp et al. 2010; Kozuka et al. 2010). Moreover, auxin transport is stimulated by red light and phyB2dependent in tomato roots (Liu et al. 2011), which might explain the difference between the WT and ppla-I-1 in coiling. Root branching is associated with polar auxin transport (Peret et al. 2009), while cotyledon expansion is dependent on the presence of phyB in white and red light, but far-red light increases cotyledons in phyB mutants (Neff & Chory 1998). Since interaction of auxin transport with phyB in physiological shade is known (Keuskamp et al. 2010; Kozuka et al. 2010), it will be interesting to investigate the influence of gravity (or absence thereof) on light responses, such as physiological shade, in suitable mutants, but it would also be useful to measure polar auxin transport in space or use auxin transport mutants in space. More research on mutants in space is warranted to understand developmental changes in microgravity, especially if successful growing of fresh food is planned as an option in a space mission. REFERENCES Brown A.H. (1993) Circumnutations: from Darwin to space flights. Plant Physiology, 101, 345–348. Buer C.S., Wasteneys G.O., Masle J. (2003) Ethylene modulates root-wave responses in Arabidopsis. Plant Physiology, 132, 1085–1096. Dutcher F.R., Hess E.L., Halstead T.W. (1994) Progress in plant research in space. Advances in Space Research, 14, 159–171. Ferl R., Wheeler R., Levine H.G., Paul A.L. (2000) Plants in space. Current Opinion in Plant Biology, 5, 258–263. Fortunati A., Piconese S., Tassone P., Ferrari S., Migliaccio F. (2008) A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heatshock factor. Journal of Experimental Botany, 59, 1363–1374. Halstead T.W., Dutcher F.R. (1987) Plants in space. Annual Review of Plant Physiology, 38, 317–345. Hoshino T., Miyamoto K., Ueda J. (2004) Automorphosis and auxin polar transport of etiolated pea

ACKNOWLEDGEMENTS This work was supported by DLR-BMBF (grant 50 WB 50WB0627 and 50WB0633). Technical assistance from Martin P€ahler, Peter Pietrzyk, Christa Ruppelt and Marianne Langer is gratefully acknowledged. Katrin Radatz and Corinna Labusch helped with quantification of the of WAICO1 data. Thanks to the many highly motivated people, a few of which can be mentioned here: R€ udiger Hartwich, Ulrich K€ ubler, Heiko Mantzsch, Volker Strobel, (ASTRIUM), Hilde Stenuit, Enno Brinkmann, Jason P. Hutton, Eric Istasse, Pierfilippo Manieri (ESA), Klaus Slenzka, Bernd Schmeyers, J€ urgen Kempf (OHB), Marianne Schuber and Dieter Seibt (DLR). SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Timeline of WAICO1 and WAICO2. Figure S2. Identification of ECs on WAICO1 and WAICO2 from their filter strips. Figure S3. Seed strip mounting tool. Figure S4. LED spectrum of light source in WAICO1 and 2. Figure S5. Experimental container and interior. Figure S6. Double centrifuge on BIOLAB model at the MultiUserCenter (MUSC-DLR, Cologne). Figure S7. All single video photos WAICO1 ‘A’ ECs (large pdf file). Figure S8. All single video photos WAICO1 ‘B’ ECs (large pdf file). Figure S9. All single video photos WAICO2 ‘A + B’ ECs (large pdf file). Figure S10. Complete astronaut photos at 12 days from WAICO1. Figure S11. Astronaut photos at 12 days from WAICO2. Figure S12. Demonstration of how in single coils the longitudinal torsion was quantified.

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