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Plant cell proliferation and growth are altered by microgravity conditions in spaceflight
ARTICLE IN PRESS Journal of Plant Physiology 167 (2010) 184–193
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Plant cell proliferation and growth are altered by microgravity conditions in spaceflight$ Isabel Matı´a a, Fernando Gonza´lez-Camacho a, Rau´l Herranz a, John Z. Kiss b, Gilbert Gasset c, Jack J.W.A. van Loon d, Roberto Marco e, Francisco Javier Medina a,n a
´gicas (CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain Centro de Investigaciones Biolo Department of Botany, Miami University, Oxford OH 45056, USA c ´ de Me´decine de Rangueil, Bˆ GSBMS, Universite´ ‘‘Paul Sabatier’’, Faculte at A2, 133 route de Narbonne, F-31062 Toulouse, France d Dutch Experiment Support Center (DESC), OCB-ACTA, Vrije Universiteit, Amsterdam, The Netherlands e Departamento de Bioquı´mica-I.I. Biome´dicas ‘‘Alberto Sols’’ (UAM-CSIC), Arzobispo Morcillo 4, E-28029 Madrid, Spain b
a r t i c l e in f o
a b s t r a c t
Article history: Received 18 June 2009 Received in revised form 24 August 2009 Accepted 25 August 2009
Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4 d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Cell cycle Microgravity Nucleolus Ribosome biogenesis Root meristem
Introduction Gravity plays a role in plant development and shaping, and its alteration induces changes in these processes (Perbal, 2001; Ferl et al., 2002). Investigation of these effects is necessary for current space exploration programs, in which the design and building of successful life support systems will be mandatory in longduration spaceflights. Abbreviations: DFC, dense fibrillar component of the nucleolus; ESA, European Space Agency; FC(s), fibrillar center(s) of the nucleolus; g, acceleration of gravity (9.8 m s 2); GC, granular component of the nucleolus; ISS, International Space Station; PBS, phosphate-buffered saline; PFA, paraformaldehyde; ROS, reactive oxygen species; RPM, random positioning machine; RT, room temperature; SIMR, stress-induced morphogenic response $ Our colleague and friend Professor Roberto Marco passed away June 27th, 2008. This paper is affectionately dedicated to his memory. n Corresponding author. Tel.: +34 91 8373112; fax: + 34 91 5360432. E-mail address: [email protected] (F. Javier Medina). 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.08.012
The influence of gravity is easily detected in plants because gravitropism affects the root and stem orientation relative to the gravity vector (Feldman, 1985). Gravitropism is related to plant growth, not only because it modulates a feature of growing plant organs but also because the gravitropic signal triggers a transduction cascade that affects the distribution of auxin (Kiss, 2000; Boonsirichai et al., 2002) and these hormones play key roles in plant growth and development (Teale et al., 2006). Most studies on the effects of gravity alteration on plant growth and development have focused on the root. The length of the primary root in microgravity, compared with 1g ground control, was found to be either similar (Volkmann et al., 1986; Perbal et al., 1987), longer (Hilaire et al., 1996; Levine and Piastuch, 2005), or even shorter (Cowles et al., 1984; Iversen et al., 1996). The causes for these discrepancies could include the multiplicity of biological systems used, the different growth conditions applied and even the influence of factors other than gravity alteration, such as the concentration of ethylene in the cabin atmosphere (Kiss et al., 1998;
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Campbell et al., 2001). In addition, it has been hypothesized that the action of microgravity might depend on the duration of exposure to microgravity and/or on the developmental stage of the plant (Claassen and Spooner, 1994). The hypothesis was confirmed experimentally in clinorotated samples (Aarrouf et al., 1999), but not with real microgravity. The processes of growth, differentiation and development affect the whole plant, but they rely on cellular mechanisms, including cell proliferation and growth, which are basic and essential functions for the cell life. It is well known that signals transduced between different plant organs are capable of activating key modulators of cell growth and cell division in a coordinated manner, in meristems; the reception of these signals and the response to them is indeed called ‘‘meristematic competence’’ (Mizukami, 2001). Furthermore, cell proliferation at the root meristem constitutes the source of cells for root growth and differentiation and the cellular basis for the developmental program of the plant (Dolan et al., 1993; Scheres et al., 2002). The alteration of environmental conditions such as gravity can modulate the activity of meristematic cells. Results obtained in space experiments have shown that the progression of the cell cycle is modified in lentil seedlings grown for 30 h in microgravity (Legue´ et al., 1996; Yu et al., 1999). A densitometric analysis of the nuclear DNA content of meristematic cells from roots grown in microgravity showed a decrease in the proportion of cells in the S phase and an increased proportion of cells in the G1 phase,
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suggesting that the G1/S transition is modulated by gravity. The mitotic index was shown to be depleted in plants grown in space (Barmicheva et al., 1989; Driss-E´cole et al., 1994); however, this result does not necessarily mean a depletion of the proliferation rate, but could be due to a decrease in the duration of mitosis or to a modification in the progression of the cell cycle. In fact, lentils grown in microgravity contain more meristematic cells in mitosis in the primary root than do ground 1g controls (Darbelley et al., 1989). Therefore, it seems clear that microgravity induces some perturbations in plant development, but it is important to consider that plants can also be sensitive to other stresses associated with the environmental conditions present in spacecrafts (Ferl et al., 2002). For that reason, the effects of altered gravity on plant development have also been observed in seedlings grown in microgravity simulators such as clinostats. An increased growth rate of primary and lateral roots and an earlier initiation of secondary roots were shown in clinorotated seedlings, leading to an increase in biomass (Driss-E´cole et al., 1994; Hilaire et al., 1996). This paper reports the results of two experiments carried out in space, consisting of seed germination, seedling growth for 4 d and chemical fixation of samples, accompanied by the corresponding controls. All of the postflight analyses were performed in the ground tissue cells of the root meristem, namely epidermis, cortex and endodermis, the three cellular layers of the cortical cylinder of the root (Scheres et al., 2002). These cells are specialized in proliferation, such that their only functional program is to grow and divide continuously
Fig. 1. Schematic representation of the scenarios used in the two space experiments reported in this paper and their corresponding controls. Note that, for the sake of clarity of the different steps, the timeline is not scaled. Each experiment comprised three phases: transport of seeds (stowage in case of ground controls) in dry condition, activation and progress of the experiment, beginning with seed hydration, continuing with incubation at constant temperature (22 1C) in order to promote growth and finishing with chemical fixation, and transport within fixative to a ground laboratory in which sample processing can proceed in normal conditions (stowage in fixative in case of ground controls). Time, temperature, sample incubation medium and light conditions are indicated for the different steps of each experiment.
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at a rate three times higher than in the central cylinder (Fujie et al., 1994). Moreover, the peak of cell divisions in roots of 6-day-old seedlings is detected at around 60 mm from the quiescent center (Beemster and Baskin, 1998). In this zone of the root tip, we analyzed a series of cellular parameters that are highly reliable indicators of the status of proliferation and growth of cells in order to assess variations in these functions prompted by a change in gravity conditions. With respect to proliferation, we measured the number of cells per millimeter in root meristematic cell files; the rate of increase of this parameter with time was called ‘‘rate of local cell production’’ (Beemster and Baskin, 1998). The concept of cell growth in meristematic cells is meant for the production of cell biomass, essentially proteins. Therefore, specifically in these cells, cell growth is determined largely by the activity of RNA polymerase I, which controls ribosomal RNA synthesis and ribosome biogenesis (Baserga, 2007). Ribosome biogenesis occurs in a well-defined nuclear territory – the nucleolus – and it has been conclusively established that, in proliferating cells, a unequivocal functional linkage exists between the rate of ribosome biogenesis and certain features of the molecular cytology of the nucleolus, namely the nucleolar size and the relative amount and distribution of nucleolar subcomponents, such as the fibrillar centers (FCs) and the granular component (GC). This has been demonstrated in many different species and cellular model systems (Shaw and Jordan, 1995; Thiry and Goessens, 1996; Hernandez-Verdun, 2006; Raska et al., 2006) and, in particular, in plants (Medina et al., 2000; Shaw and Doonan, 2005; Sa´ez-Va´squez and Medina, 2008), always using cellular models characterized by high cell proliferating activity. Furthermore, regulation of ribosome production is controlled by a subset of nucleolar proteins whose activity is, in turn, controlled by factors regulating cell cycle progression (Klein and Grummt, 1999; Sa´ez-Va´squez and Medina, 2008). The major nucleolar protein is nucleolin, a protein conserved in animals, plants and yeast, whose levels are correlated with the rate of functional activity of the nucleolus in exponentially growing cells (Ginisty et al., 1999; Sa´ez-Va´squez and Medina, 2008). The analysis of these parameters in root meristematic cells of samples grown in space and in the corresponding ground controls has led us to define the alterations induced by microgravity on cell growth and proliferation. We found a striking decoupling of these two processes that are strictly coordinated in ground conditions.
Materials and methods The results presented in this paper correspond to an experiment performed on the ISS in the course of the Spanish ‘‘Cervantes’’ Soyuz mission, which took place in October 2003 (van Loon et al., 2007). Samples obtained from this experiment were complemented with some others, coming from the STS-84 ‘‘Atlantis’’ Space Shuttle mission of NASA from May 1997, in which Arabidopsis seedlings were germinated and grown in space in similar conditions (Kiss et al., 1999). In addition, a ground control experiment in a random positioning machine (RPM), a reliable simulator of microgravity (van Loon, 2007b), was performed under the same conditions, and using the same hardware as in the ISS experiment. An overall schematic view of the scenarios used in the different experiments is presented in Fig. 1. ISS experiment Seeds of Arabidopsis thaliana (L.), Heynh., ecotype ‘‘Columbia’’ (Col-0), which had been stored for at least three weeks at 4 1C for vernalization, were sterilized in 1.25% (v/v) sodium hypochlorite
and 1% (v/v) Triton X-100 for 10 min and dried onto a sheet of filter paper (Whatman CHR1), 5 3 cm (approx. 90 seeds per sheet). They were prepared to be sent to space and then to germinate, grow and be fixed in-flight, using a device prepared and assembled according to the ‘‘Berlingot-Ampoule’’ concept, first introduced by the ‘‘Groupement Scientifique en Biologie et Me´decine Spatiales’’ (GSBMS, Toulouse, France) (Tixador et al., 1981). Briefly, two sets of 10 ampoules, 30 mL each, one set containing culture medium (Murashige and Skoog’s, Duchefa) and the other set containing a fixative solution of 8% (v/v) paraformaldehyde (PFA, EM grade, EMS, Fort Washington PA, USA) in PBS, were introduced in a ‘‘Berlingot’’, that is, a double-wall plastic bag, together with seeds and filter paper and then sealed. Two ‘‘Berlingots’’ were then introduced into hermetic Biorack type I/E biocontainers supplied with the device known as motorized ampoule breaker assembly (MAMBA), a technical development of Dutch Space B.V. (Leiden, The Netherlands) in order for the semi-automatic breakage of ampoules. A detailed description of this hardware, including graphic documentation, was published previously (Matı´a et al., 2007). Once in the ISS, the experiment was activated by breaking the first set of ampoules and leaving the culture medium to wet the filter paper. This promoted germination of seeds. At this moment, biocontainers were transferred to an incubator at a constant temperature of 22 (70.5) 1C. After 4 d, the second set of ampoules was broken, releasing the fixative solution, which, mixed with the same amount of culture medium already present within the ‘‘Berlingot’’, gave a paraformaldehyde (PFA) concentration of 4%. All operations were performed semi-automatically, only by activating an electric motor within each biocontainer. Therefore, biocontainers remained hermetically closed throughout all the process, so that seedling germination and growth occurred in darkness and the available air was that initially contained in each Biorack container (the ‘‘Berlingot’’ plastic walls are permeable to air). The experiment materials returned back to the Earth on the same day of sample fixation. The temperature of the biocontainers progressively rose from 22 to 27 1C during the journey (approximately 8 h). Once on Earth, they were stored in an active refrigerated container at 4 1C, where they were transported from the landing site in Kazakhstan to the ‘‘Star City’’ Russian Space Complex, near Moscow (this transport took another period of approximately10 h). In this location, the experimental materials were received by the investigators. Biocontainers were immediately opened, and samples were photographed and transferred to PBS for washing (3 10 min) and partially dehydrated in an ethanol series until 70% ethanol; in this medium they were transported to Madrid, Spain, for final processing for microscopy and immunocytochemistry, including full dehydration and embedding in LR White resin (London Resin Co., UK).
Space shuttle (STS-84 mission) experiment This experiment was carried out in the ESA payload ‘‘Biorack’’, flown in the American Space Shuttle ‘‘Atlantis’’ (Kiss et al., 1999). In this experiment, Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija Ws) seeds germinated and seedlings grew in orbit at 22 1C for 90 h in the darkness. A red light pulse of 10 min was given 24 h after seed soaking to stimulate germination. Fixation was initiated in-flight with 4% (v/v) glutaraldehyde in 100 mmol/L phosphate buffer and extended for 5.5 d, until samples were recovered on ground, where processing continued with washing in buffer, dehydration in ethanol series and embedding in Quetol 651 resin (Guisinger and Kiss, 1999; Kiss et al., 1999). A control experiment in-flight, using a 1g centrifuge, was performed under the same conditions.
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Random positioning machine (RPM) experiment This experiment was performed in the RPM located in the Dutch Experiment Support Center (DESC), Vrije Universiteit, Amsterdam, The Netherlands. This machine does not remove gravity, but its action becomes omnilateral instead of unilateral, resulting in the effect that plants are not capable of responding to a definite gravity vector (van Loon, 2007b). The biological material, hardware used, times and temperatures of growth and sample processing were an exact replica of the ISS experiment. The rotational velocity of the RPM frames was randomized with a maximum of 7601 s 1; the interval was set at random. The samples were positioned in the center of the inner frame, and the largest radius was, at maximum, 5 cm to the outermost biocontainer. This resulted in a maximum residual gravity of less than 10 4g (van Loon, 2007b).
Control 1g experiments Ground 1g control experiments were performed in our laboratory for the three experiments described above, namely that of the ISS, that of the Space Shuttle and that of the RPM. In these control experiments the biological material, hardware used, times and temperatures of growth and sample processing were the same or exact replicas as in the reference experiments. In the case of the RPM, control biocontainers were positioned in the outer static frames of the instrument.
Microscopy, immunocytochemistry and quantification From the materials embedded in resin, semithin sections, 2 mm thick, were cut and observed unstained under a Leica DM2500 microscope equipped with phase contrast. Images were digitally recorded with a Leica DFC320 CCD camera. For ultrastructural studies, ultrathin sections were mounted on Formvar-coated nickel grids stained with uranyl acetate and lead citrate and observed in a Jeol 1230 electron microscope, operating at 100 kV. For immunogold labeling, ultrathin sections were incubated with an anti-nucleolin antibody (kindly supplied by Dr. J. Sa´ez-Va´squez, CNRS, Perpignan, France) diluted 1:100, for 90 min at room temperature (RT), washed repeatedly and incubated with goat anti-rabbit IgG secondary antibody coupled with 10 nm colloidal gold particles (Sigma-Aldrich, St Louis MO, USA), diluted 1:50, for
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1 h, at RT. Prior to observation, grids were counterstained with uranyl acetate, either alone or followed by lead citrate. Quantitative measurements (seedling and root length, number of cells per mm in root cell files, cross-sectional area of nucleoli, proportion of nucleolar granular component (GC) and density of gold particles) were carried out on digital images using the quantitation software ‘‘QWin Standard’’ (Leica Microsystems). Measurements of cell parameters were performed in a zone of 50 730 mm above the quiescent center (approx. 100730 mm from the root tip), corresponding to the highest rate of cell division, according to the available literature (Beemster and Baskin, 1998). In this zone, epidermal, cortical and endodermal cells were selected. The number of samples measured was: 20 seedlings for seedling length, 10 roots for number of cells per mm, 60 nucleoli (light microscopy) for cross-sectional area and 15 nucleoli (electron microscopy) for GC proportion and density of gold particles. Statistical analysis of data was performed using t SPSS 13.0 software. Description of quantitative variables was performed using means and standard deviations after checking normality with the Kolmogorov–Smirnov test. Mean values were compared using the Student’s t-test for independent samples; differences were considered significant for a bilateral a value lower than 0.05.
Results Seeds germinated in either real or simulated microgravity showed a high rate of germination (more than 75%), comparable with the rate obtained in ground 1g control conditions. In all cases, germination and growth for 4 d in darkness produced etiolated seedlings, lacking any green part and showing a highly developed hypocotyl (Fig. 2a). As expected, growth orientation of roots and hypocotyls in seedlings from both real and simulated microgravity experiments was random, whereas seedlings from 1g controls appeared oriented with respect to the gravity vector. Seedlings grown in space and grown in the random positioning machine (RPM) showed a longer size than those of the ground 1g control, and the differences were significant (Fig. 2). However, no significant differences in length were found between the flight samples and the RPM samples (Fig. 2b). The increase in length of the seedlings grown in either real or simulated microgravity was due to an increased length of both the root and the hypocotyl (Fig. 2a). Root length was significantly longer in the flight samples and the RPM samples, but the difference with respect to the
Fig. 2. a: Seedlings of Arabidopsis thaliana grown in space (flight, left), in simulated microgravity in the Random Positioning Machine (RPM, center) and in control 1g ground conditions (right). Growth in darkness produced etiolated seedlings with a very developed hypocotyl. Compared at the same magnification, a longer size of seedlings grown in microgravity (either real or simulated) stands out clearly. b, c: Quantitative measurement of the seedling length (b) and the root length (c) from the spaceflight experiment and its associated ground control experiments. Mean and standard deviation values are graphically represented. Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (p o 0.05). N = 20 seedlings for each condition.
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Fig. 3. Semithin (2 mm thick) sections of the root distal part from samples grown under the different conditions, observed under the phase-contrast microscope. The difference in shape and size between the larger and more regular cells of the cortical cylinder (CoC) with respect to the longer and narrower cells of the central cylinder (CeC) is clearly appreciated. Cells of the cortical cylinder, organized as the files called epidermis (Ep), cortex (Co) and endodermis (En), constitute the ground tissue root meristem. There are more of these cells in each file, and they are smaller, in the microgravity-grown samples (flight and RPM) than in the ground 1g control. QC: Quiescent center. Col: Columella. LRC: Lateral root cap.
Fig. 4. Estimation of the cell proliferation rate in the cortical root meristematic cell files, by counting the number of cells per millimeter in each file. Ten roots (root meristems) were used for each condition. Mean and standard deviation values are graphically represented. Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (p o0.05).
ground 1g control was less than the difference recorded for whole seedlings (Fig. 2c). Semithin sections of the root meristem obtained from all samples, observed under phase-contrast microscopy, showed the files of cells corresponding to both the central and the cortical cylinder. At the root tip, quiescent center and columella cells were visible (Fig. 3). In the three files of the cortical cylinder (epidermis,
cortex and endodermis), and in a zone of 50 730 mm above the quiescent center (approx. 100 730 mm from the root tip) we counted the number of cells per mm. This number was significantly higher in the flight and the RPM samples compared to the ground 1g control; no significant difference was found between the flight and the RPM samples (Fig. 4). Consequently, samples grown in either real or simulated microgravity showed a higher cell proliferation rate (notice that, in addition to containing more cells per mm, roots were longer in these conditions; see Fig. 2) and the cells were shorter than in control conditions. In addition to the quantitative demonstration, this feature was clearly observed in microscopic images (Fig. 3). Estimations of the alterations in cell growth induced by microgravity were based on certain morphological and morphometrical features of the nucleolus that have been demonstrated to be functionally associated with the rate of ribosome biogenesis in proliferating cells. The features are the nucleolar size, the relative proportion and distribution of nucleolar subcomponents and the levels of the nucleolar protein nucleolin. To investigate these features we used samples fixed in glutaraldehyde, as well as those fixed in PFA, together with their respective ground 1g controls. Fixation in glutaraldehyde was necessary for clear discrimination of nucleolar subcomponents; fixation in PFA was necessary for immunogold localization of nucleolin. Nevertheless, comparison of the overall nucleolar ultrastructure in the two sets of experiments showed that, for the purpose of our studies, samples were comparable (compare Fig. 5a–c with Fig. 5d–f and Fig. 7). In samples fixed with glutaraldehyde, nucleolar subcomponents were clearly discernible (Fig. 5a–c). In all cases, the nucleolar ultrastructure corresponded to that of a proliferating cell actively engaged in the production of ribosomes, showing
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Fig. 5. a–c: Ultrastructure of nucleoli from root meristematic cortical cells fixed in glutaraldehyde in space and its associated ground control experiment. a: Sample grown in space (flight lg); b: sample grown in the 1g centrifuge, on board of the spacecraft (flight 1g), and c: control sample, grown on Earth (ground 1g). Notice the difference in size between the three samples. The three basic nucleolar subcomponents are clearly distinguished, namely fibrillar centers (arrows), dense fibrillar component (DFC) and granular component (GC). Fibrillar centers are larger and less numerous in the flight lg sample, whereas granular component is more abundant in both samples grown at 1g, and this component appears interspersed with territories of dense fibrillar component. d–f: Ultrastructure of nucleoli from root meristematic cortical cells fixed in paraformaldehyde in space and its associated ground control experiments. d: sample grown in space (flight); e: sample grown in the random positioning machine (RPM), under simulated microgravity, and f: control sample, grown on Earth (ground 1g). Notice the difference in size between the three samples. Discrimination of nucleolar subcomponents is more difficult than with glutaraldehyde fixation. However, fibrillar centers are clearly distinguished (arrows) and the granular component (GC) can be differentiated from the dense fibrillar component (DFC), mostly in the peripheral zones. The pattern of number and size of fibrillar centers is similar to that described for glutaraldehyde-fixed samples. Scale bars indicate 1 lm.
features such as an abundant GC (which accounts for approx. 50% of the nucleolar volume) and small FCs of the homogeneous type. However, the nucleolar size was significantly smaller in samples grown in space under microgravity than in samples grown in the 1g centrifuge in space and in the ground 1g control. The nucleolar size in these two latter samples did not show significant differences (Fig. 6a). Considering the amount and distribution of nucleolar components, the microgravity-grown samples showed the GC occupying a peripheral position with respect to the dense fibrillar component (DFC) and FCs, which occupied a central position in the section of the nucleolus (Fig. 5a). In contrast, in the 1g samples (either centrifuge or ground) the GC appeared intermingled with masses of the DFC, which were observed interspersed in the nucleolar section, with small FCs in their interior (Fig. 5b–c). Quantitatively, the GC accounted for 54–57% of the nucleolar volume in 1g samples, whereas it was only around
45% of nucleoli of cells from samples grown in microgravity (Fig. 6a). Taking into account the difference in size between nucleoli from the two types of samples, the amount of nucleolar GC was severely depleted by growth in spaceflight under microgravity relative to the 1g gravity conditions. In fact, practically all of the differences in size between nucleoli from cells grown in various gravity conditions were accounted for by the GC, with the fibrillar components (DFC and FCs) showing a minimal contribution (Fig. 6a). The results of PFA fixation were in agreement with those obtained with glutaraldehyde fixation. The differences were a moderate shrinkage of the organelle, which was a consequence of the reduction in the quality of fixation (Lerı´a et al., 2004), which affected equally to all the samples, whatever the growth conditions, and, as previously mentioned, a difficult discrimination of subnucleolar components, mostly the GC from the DFC,
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Fig. 6. Nucleolar size, measured as cross-sectional area, in root meristematic cortical cells fixed in glutaraldehyde (a) and in paraformaldehyde (b) in space and its associated control experiments. Absolute values are lower for paraformaldehyde- than those recorded for glutaraldehyde-fixed samples, due to poorer quality of fixation. The reduction affected equally to all conditions. Mean and standard deviation values are graphically represented. Dotted areas in the bars indicate the proportion occupied by granular component (GC). Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (p o 0.05). N = 60 nucleoli for each condition, for nucleolar area; N =15 nucleoli for each condition, for GC proportion.
Fig. 7. Immunogold electron microscopic localization of the nucleolar protein nucleolin in root meristematic cortical cells fixed in paraformaldehyde in space, and its associated ground control experiments. From left to right, sample grown in space (flight), sample grown in the random positioning machine (RPM), under simulated microgravity, and control sample, grown on Earth (ground 1g). In general, gold particles appear in the nucleolus, preferentially located in the dense fibrillar component (DFC) surrounding fibrillar centers (arrows). This localization is more conspicuous in the ground 1g control, whereas in samples grown in microgravity (real or simulated), gold particles are more dispersed throughout the nucleolar area. Fibrillar centers themselves, as well as the granular component (GC) appear devoid of labeling. Scale bars indicate 1 mm.
and mainly in the boundary zones (Fig. 5d–f and Fig. 7). In this case, the nucleoli were significantly smaller in the flight and the RPM samples (i.e. those grown under microgravity, either real or simulated) relative to the ground 1g control (Fig. 6b). For subnucleolar components, FCs appeared smaller and more numerous in the ground 1g control (Fig. 5f). Finally, the quantitative immunolocalization of the major nucleolar protein, nucleolin, was used as an additional indicator of the rate of ribosome biogenesis. Electron microscopic immunolocalization is advantageous since it not only allows the
estimation of the levels of the protein, but also its intranucleolar distribution, which is a feature with functional significance (Pontvianne et al., 2007). Nucleolin in all cases was located in the DFC of the nucleolus, near FCs, where it was previously described to be located in active nucleoli of plant meristematic cells. This localization was more evident in the ground 1g control than in the flight and RPM samples, in which the gold particles appeared to be more dispersed throughout the nucleolar area, resembling the pattern observed in low-active nucleoli (Fig. 7). Quantitatively, ground 1g control cell nucleoli showed a
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Fig. 8. Quantitative measurement of the nucleolin levels, estimated by the density of gold particles (no. of gold particles per mm2), after immunolabeling, in root meristematic cortical cells from the spaceflight experiment and its associated ground control experiments. Mean and standard deviation values are graphically represented. Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (po 0.05). N = 15 nucleoli for each condition.
significantly higher density of labeling than nucleoli from either flight or RPM-grown samples, which did not show significant differences between them (Fig. 8). This was an additional confirmation of the depletion of the rate of ribosome biogenesis occurring in cells grown in microgravity, either real (spaceflight) or simulated (RPM). Remarkably, for all measurements performed, no significant differences were found between samples grown in spaceflight and in the RPM. Measurements performed in the ground 1g control samples and in samples grown in the 1g centrifuge during spaceflight were also not significantly different.
Discussion The spaceflight environment comprises a collection of conditions, such as microgravity, radiation and other factors that can influence life processes. The differentiation of microgravity effects from the other environmental factors that are present in space is the main reason to apply an in-flight 1g centrifuge (providing all space factors but microgravity) and to perform ground control experiments using devices that simulate microgravity, such as RPM (providing microgravity, but no other space factors), whenever possible (van Loon, 2007a). According to the results obtained in this study and comparing the measured parameters in the different environmental conditions tested, we conclude that microgravity is the most important source of alteration. This conclusion comes from the comparison of the results obtained in the spaceflight experiment (real microgravity) with those obtained in the RPM (simulated microgravity), which did differ significantly. The conclusion is also supported by the comparison of the results obtained from the flight 1g experiment and the ground 1g experiment, which were statistically similar. Additionally, RPM was confirmed in our work as a good and reliable simulator of microgravity, in line with previous published results using other parameters, such as the position of amyloplasts in columella cells, or the changes in shape of attached cells in culture (Kraft et al., 2000; van Loon, 2007b). The enormous advances in recent years producing knowledge of genomic data, in particular in the model plant species A. thaliana, has
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led to the understanding that a sizeable number of genes modify their expression pattern in response to changes in the environmental gravity conditions (Moseyko et al., 2002; Centis-Aubay et al., 2003; Kimbrough et al., 2004; Martzivanou et al., 2006; Salmi and Roux, 2008). In some cases, the gravity alteration consisted of the reorientation of the growth direction, sometimes accompanied by a simple mechanical stimulation (Moseyko et al., 2002; Kimbrough et al., 2004). In other cases, ground-based microgravity simulators (clinostat or RPM) were used to induce this alteration (Centis-Aubay et al., 2003; Martzivanou et al., 2006), and in the last case, the analysis was performed on fern spores germinated in spaceflight (Salmi and Roux, 2008). The identification of altered genes reveals a heterogeneous group comprising transcription factors, modulators of cell wall features, elements of early signaling cascades and signal transduction pathways, hormone-responsive elements and other genes of unknown function. Several of these genes also appear altered in plants affected by known environmental stresses (dehydration, cold, pathogen response). Some general conclusions have been reached from these genomic studies, such as the identification of the gravity alteration as an actual stress condition for the plant, the existence of a mechanism of signaling, and a signal transduction cascade as a response to the alteration and the involvement of hormones, and specifically auxin in this response. However, these types of analysis have supplied few data on the cellular mechanisms altered by the gravity modification. In particular, changes in basic cellular processes influencing plant growth and development are scarcely documented in these studies, even though some of the genes modified in their expression may influence these cellular processes. Therefore, the genomic approach, while of great importance and interest, should be supplemented by other experimental approaches providing complementary information by means of the use of additional parameters, in order to get the full picture of the functional alterations induced by the environmental change. Our results clearly show that growth in the microgravity environment of spaceflight produces substantial alterations in root meristematic cell growth and proliferation. Cell proliferation appears enhanced, whereas the cell size and the rate of production of ribosomes (both taken as indicators of cell growth) appear depleted. With respect to ribosome biogenesis, all morphological and morphometrical features of nucleoli, together with the immunocytochemical estimation of the levels of the nucleolar protein nucleolin, indicated a depletion in the rate of pre-rRNA transcription and processing, according to the morpho-functional models, previously published and generally agreed upon (Shaw and Jordan, 1995; Medina et al., 2000). A lowering in the rate of ribosome biogenesis was previously detected in our laboratory in root meristematic cells from clinorotated samples (Sobol et al., 2005; 2006), but it was not put in relation to other cellular processes. Cell proliferation and cell growth, which are closely associated and interdependent processes in this cell type under ground gravity conditions, appear divergent in the microgravity environment. Our interpretation of this divergence is that the cell cycle is altered by the change in gravity conditions. It is known that the major part of ribosome production (and consequently, of cell growth) in cycling cells occurs in G2 as a preparation for mitosis (Sa´ez-Va´squez and Medina, 2008). A shortening of the G2 phase could result in a reduction in cell growth/ribosome biogenesis and in an acceleration of cell division, producing a greater number of shorter cells, i.e. the experimental result we obtained in the spaceflight and RPM tests. Checkpoints for cell size occur indeed just in the G2 stage and in the G2-mitosis transition (Mizukami, 2001; Sugimoto-Shirasu and Roberts, 2003; Inze´ and De Veylder, 2006). Changes in cell cycle duration have been described in young seedlings grown in spaceflight (Perbal and Driss-E´cole, 1994; Yu et al., 1999). In these experiments, carried out in the Spacelab IML-1
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and IML-2 missions, lentil seedlings were grown for 28–29 h, i.e. the time necessary to complete the first cell cycle after germination. The percentages of the different cell cycle phases (G1, S, G2, M) were determined after Feulgen staining. In the two experiments, samples grown in space were enriched in earlier phases with respect to 1g controls, and the authors concluded that the cell cycle was slowed down in microgravity. However, this conclusion does not take into account a possible different effect of microgravity depending on the time of exposure and/or on the phase of development of the plant or seedling (Claassen and Spooner, 1994; Aarrouf et al., 1999). In fact, this time of growth under microgravity conditions was quite different in these experiments (28–29 h) and in the experiment reported in the present paper (4 d). The origin of the observed alterations in cell proliferation and cell growth in a weightless environment could be either the transduction of the mechano-signal sensed by columella cells and responsible for gravitropism, or the perception of independent signals by cells not apparently specialized in gravity sensing (Kordyum, 1997). In favor of the first possibility is the fact that the transduction of the gravitropic signal finally results in the change of the distribution of auxin streams in the root, producing lateral gradients of this hormone (Boonsirichai et al., 2002). It is known that auxin plays a role in the regulation of cell growth and cell proliferation by molecular mechanisms that are beginning to be understood (Himanen et al., 2002; David et al., 2007). Studies carried out on cultured animal cells in weightlessness (which obviously lack mechano-receptors comparable to those of statocytes), showing alterations in lymphocyte proliferation (Cogoli and Cogoli-Greuter, 1997), melanoma growth and tumorigenicity (Dai et al., 2007), or osteogenesis of bone marrow stem cells (Taga et al., 2006), among others, would support the second alternative. The alteration of basic and essential cellular processes in the root meristem as a consequence of gravity changes indicate that this environmental modification can be considered as a real stress for the plant that may have consequences in plant morphogenesis and development. Furthermore, it could be hypothesized that plant responses described for other abiotic stress conditions, such as the so-called ‘‘stress-induced morphogenic response’’ (SIMR) (Potters et al., 2007), could also be found as a consequence of the growth in a microgravity environment. Certainly, the alterations that we have observed do not fit exactly with the model of SIMR, but similar parameters (cell growth, cell proliferation and seedling morphology) are affected and the response observed after the gravitational stress is indeed a morphogenic response. Interestingly, the cellular and molecular mechanisms associated with SIMR and triggering its effects are auxin transport and/or metabolism, cell cycle regulation and production of reactive oxygen species (ROS), a class of molecules with high oxidative capacity (Potters et al., 2007). In relation to these molecules, a group of genes belonging to the functional category of the oxidative burst, related to the plant defense mechanisms, was found to be overexpressed after gravitational stimulation (Kimbrough et al., 2004). It would be interesting to analyze the presence of ROS (and their possible induction) in plants grown under microgravity or altered gravity conditions. In conclusion, microgravity conditions during spaceflight produce stress for seed germination and seedling growth which, at the cellular level, results in the divergence of cell proliferation and cell growth in root meristematic cells. These two processes are strictly related to each other in this cellular type in normal ground gravity conditions. Even though the technical limitations of the spaceflight experiment did not allow us to perform additional experiments, we can hypothesize that shortening of one or more phases of the cell cycle, produced by the alteration of at least some checkpoints responsible for their regulation, could
account for the observed effects. Experiments involving the analysis of the expression of key factors in the regulation of cell cycle progression at the level of these checkpoints, using more points of seedling development for data recording, are currently in progress (Matı´a et al., 2009) and should contribute to clarification of this important problem.
Acknowledgements The authors thank the European Space Agency (ESA) astronaut Pedro Duque (ISS experiments) and the astronauts of the STS-84 Mission (Space Shuttle experiments) who performed the in-flight operations of the experiments reported in this paper. We also thank Mrs. Mercedes Carnota (CIB-CSIC, Madrid, Spain) and Mrs. Brigitte Eche (GSBMS, Toulouse, France) for excellent technical assistance in the ground experiments. The generous gift of anti-nucleolin antibody by Dr. Julio Sa´ez-Va´squez (CNRS, Perpignan, France) is gratefully acknowledged, as well as the supply of MAMBAs by Dutch Space B.V. (Leiden, The Netherlands). The ESA personnel at ESTEC (Noordwijk, The Netherlands) were of great help in the preparation of the ISS Mission; in particular, we would like to thank Mr. Jesu´s Jime´nez, Ms. Natalie Pottier and Mr. Fabrizio Festa, as well as Dr. Didier Chaput (CNES, Toulouse, France). This work was supported by grants from the Spanish ‘‘Plan Nacional de I+D+i’’, Refs. nos. ESP2001-4522-PE and ESP2006-13600-C02-02, and the use of RPM, by Grant no. MG-057 (SRON, NL). References Aarrouf J, Darvelley N, Demandre C, Razafindranboa N, Perbal G. Effect of horizontal clinorotation on the root system development and on lipid breakdown in Rapeseed (Brassica napus) seedlings. Plant Cell Physiol 1999;40:396–405. Barmicheva EM, Grif VG, Tairbekov MG. Growth and structure of cells in the maize root apex under space flight conditions. Cytology 1989;31:1324–8. Baserga R. Is cell size important?. Cell Cycle 2007;6:814–6. Beemster GTS, Baskin TI. Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol 1998;116:1515–26. Boonsirichai K, Guan C, Chen R, Masson PH. Root gravitropism: an experimental tool to investigate basic cellular and molecular processes underlying mechanosensing and signal transmission in plants. Annu Rev Plant Biol 2002;53: 421–447. Campbell WF, Salisbury FB, Bugbee B, Klassen S, Naegle E, Strickland DT, et al. Comparative floral development of Mir-grown and ethylene-treated, earthgrown super dwarf wheat. J Plant Physiol 2001;158:1051–60. Centis-Aubay S, Gasset G, Mazars C, Ranjeva R, Graziana A. Changes in gravitational forces induce modifications of gene expression in A. thaliana seedlings. Planta 2003;218:179–85. Claassen DE, Spooner BS. Impact of altered gravity on aspects of cell biology. Int Rev Cytol 1994;156:301–73. Cogoli A, Cogoli-Greuter M. Activation and proliferation of lymphocytes and other mammalian cells in microgravity. Adv Space Biol Med 1997;6:33–79. Cowles JR, Scheld HW, Lemay R, Peterson C. Growth and lignification in seedlings exposed to eight days of microgravity. Ann Bot 1984;54:33–48. Dai ZQ, Wang R, Ling SK, Wan YM, Li YH. Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Proliferation 2007;40:671–84. Darbelley N, Driss-E´cole D, Perbal G. Elongation and mitotic activity of cortical cells in lentil roots grown in microgravity. Plant Physiol Biochem 1989;27: 341–347. David KM, Couch D, Braun N, Brown S, Grosclaude J, Perrot-Rechenmann C. The auxin-binding protein 1 is essential for the control of cell cycle. Plant J 2007;50:197–206. Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, et al. Cellular organisation of the Arabidopsis thaliana root. Development 1993;119:71–84. Driss-E´cole D, Schoevaert D, Noin M, Perbal G. Densitometric analysis of nuclear DNA content in lentil roots grown in space. Biol Cell 1994;81:59–64. Feldman LJ. Root gravitropism. Physiol Plant 1985;65:341–4. Ferl R, Wheeler R, Levine HG, Paul AL. Plants in space. Curr Opin Plant Biol 2002;5:258–63. Fujie M, Kuroiwa H, Suzuki T, Kawano S, Kuroiwa T. Organelle DNA Synthesis in the quiescent centre of Arabidopsis thaliana (Col.). J Exp Bot 1994;44:689–93. Ginisty H, Sicard H, Roger B, Bouvet P. Structure and functions of nucleolin. J Cell Sci 1999;112:761–72.
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Plant cell proliferation and growth are altered by microgravity conditions in spaceflight$ Isabel Matı´a a, Fernando Gonza´lez-Camacho a, Rau´l Herranz a, John Z. Kiss b, Gilbert Gasset c, Jack J.W.A. van Loon d, Roberto Marco e, Francisco Javier Medina a,n a
´gicas (CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain Centro de Investigaciones Biolo Department of Botany, Miami University, Oxford OH 45056, USA c ´ de Me´decine de Rangueil, Bˆ GSBMS, Universite´ ‘‘Paul Sabatier’’, Faculte at A2, 133 route de Narbonne, F-31062 Toulouse, France d Dutch Experiment Support Center (DESC), OCB-ACTA, Vrije Universiteit, Amsterdam, The Netherlands e Departamento de Bioquı´mica-I.I. Biome´dicas ‘‘Alberto Sols’’ (UAM-CSIC), Arzobispo Morcillo 4, E-28029 Madrid, Spain b
a r t i c l e in f o
a b s t r a c t
Article history: Received 18 June 2009 Received in revised form 24 August 2009 Accepted 25 August 2009
Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4 d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Cell cycle Microgravity Nucleolus Ribosome biogenesis Root meristem
Introduction Gravity plays a role in plant development and shaping, and its alteration induces changes in these processes (Perbal, 2001; Ferl et al., 2002). Investigation of these effects is necessary for current space exploration programs, in which the design and building of successful life support systems will be mandatory in longduration spaceflights. Abbreviations: DFC, dense fibrillar component of the nucleolus; ESA, European Space Agency; FC(s), fibrillar center(s) of the nucleolus; g, acceleration of gravity (9.8 m s 2); GC, granular component of the nucleolus; ISS, International Space Station; PBS, phosphate-buffered saline; PFA, paraformaldehyde; ROS, reactive oxygen species; RPM, random positioning machine; RT, room temperature; SIMR, stress-induced morphogenic response $ Our colleague and friend Professor Roberto Marco passed away June 27th, 2008. This paper is affectionately dedicated to his memory. n Corresponding author. Tel.: +34 91 8373112; fax: + 34 91 5360432. E-mail address: [email protected] (F. Javier Medina). 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.08.012
The influence of gravity is easily detected in plants because gravitropism affects the root and stem orientation relative to the gravity vector (Feldman, 1985). Gravitropism is related to plant growth, not only because it modulates a feature of growing plant organs but also because the gravitropic signal triggers a transduction cascade that affects the distribution of auxin (Kiss, 2000; Boonsirichai et al., 2002) and these hormones play key roles in plant growth and development (Teale et al., 2006). Most studies on the effects of gravity alteration on plant growth and development have focused on the root. The length of the primary root in microgravity, compared with 1g ground control, was found to be either similar (Volkmann et al., 1986; Perbal et al., 1987), longer (Hilaire et al., 1996; Levine and Piastuch, 2005), or even shorter (Cowles et al., 1984; Iversen et al., 1996). The causes for these discrepancies could include the multiplicity of biological systems used, the different growth conditions applied and even the influence of factors other than gravity alteration, such as the concentration of ethylene in the cabin atmosphere (Kiss et al., 1998;
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Campbell et al., 2001). In addition, it has been hypothesized that the action of microgravity might depend on the duration of exposure to microgravity and/or on the developmental stage of the plant (Claassen and Spooner, 1994). The hypothesis was confirmed experimentally in clinorotated samples (Aarrouf et al., 1999), but not with real microgravity. The processes of growth, differentiation and development affect the whole plant, but they rely on cellular mechanisms, including cell proliferation and growth, which are basic and essential functions for the cell life. It is well known that signals transduced between different plant organs are capable of activating key modulators of cell growth and cell division in a coordinated manner, in meristems; the reception of these signals and the response to them is indeed called ‘‘meristematic competence’’ (Mizukami, 2001). Furthermore, cell proliferation at the root meristem constitutes the source of cells for root growth and differentiation and the cellular basis for the developmental program of the plant (Dolan et al., 1993; Scheres et al., 2002). The alteration of environmental conditions such as gravity can modulate the activity of meristematic cells. Results obtained in space experiments have shown that the progression of the cell cycle is modified in lentil seedlings grown for 30 h in microgravity (Legue´ et al., 1996; Yu et al., 1999). A densitometric analysis of the nuclear DNA content of meristematic cells from roots grown in microgravity showed a decrease in the proportion of cells in the S phase and an increased proportion of cells in the G1 phase,
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suggesting that the G1/S transition is modulated by gravity. The mitotic index was shown to be depleted in plants grown in space (Barmicheva et al., 1989; Driss-E´cole et al., 1994); however, this result does not necessarily mean a depletion of the proliferation rate, but could be due to a decrease in the duration of mitosis or to a modification in the progression of the cell cycle. In fact, lentils grown in microgravity contain more meristematic cells in mitosis in the primary root than do ground 1g controls (Darbelley et al., 1989). Therefore, it seems clear that microgravity induces some perturbations in plant development, but it is important to consider that plants can also be sensitive to other stresses associated with the environmental conditions present in spacecrafts (Ferl et al., 2002). For that reason, the effects of altered gravity on plant development have also been observed in seedlings grown in microgravity simulators such as clinostats. An increased growth rate of primary and lateral roots and an earlier initiation of secondary roots were shown in clinorotated seedlings, leading to an increase in biomass (Driss-E´cole et al., 1994; Hilaire et al., 1996). This paper reports the results of two experiments carried out in space, consisting of seed germination, seedling growth for 4 d and chemical fixation of samples, accompanied by the corresponding controls. All of the postflight analyses were performed in the ground tissue cells of the root meristem, namely epidermis, cortex and endodermis, the three cellular layers of the cortical cylinder of the root (Scheres et al., 2002). These cells are specialized in proliferation, such that their only functional program is to grow and divide continuously
Fig. 1. Schematic representation of the scenarios used in the two space experiments reported in this paper and their corresponding controls. Note that, for the sake of clarity of the different steps, the timeline is not scaled. Each experiment comprised three phases: transport of seeds (stowage in case of ground controls) in dry condition, activation and progress of the experiment, beginning with seed hydration, continuing with incubation at constant temperature (22 1C) in order to promote growth and finishing with chemical fixation, and transport within fixative to a ground laboratory in which sample processing can proceed in normal conditions (stowage in fixative in case of ground controls). Time, temperature, sample incubation medium and light conditions are indicated for the different steps of each experiment.
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at a rate three times higher than in the central cylinder (Fujie et al., 1994). Moreover, the peak of cell divisions in roots of 6-day-old seedlings is detected at around 60 mm from the quiescent center (Beemster and Baskin, 1998). In this zone of the root tip, we analyzed a series of cellular parameters that are highly reliable indicators of the status of proliferation and growth of cells in order to assess variations in these functions prompted by a change in gravity conditions. With respect to proliferation, we measured the number of cells per millimeter in root meristematic cell files; the rate of increase of this parameter with time was called ‘‘rate of local cell production’’ (Beemster and Baskin, 1998). The concept of cell growth in meristematic cells is meant for the production of cell biomass, essentially proteins. Therefore, specifically in these cells, cell growth is determined largely by the activity of RNA polymerase I, which controls ribosomal RNA synthesis and ribosome biogenesis (Baserga, 2007). Ribosome biogenesis occurs in a well-defined nuclear territory – the nucleolus – and it has been conclusively established that, in proliferating cells, a unequivocal functional linkage exists between the rate of ribosome biogenesis and certain features of the molecular cytology of the nucleolus, namely the nucleolar size and the relative amount and distribution of nucleolar subcomponents, such as the fibrillar centers (FCs) and the granular component (GC). This has been demonstrated in many different species and cellular model systems (Shaw and Jordan, 1995; Thiry and Goessens, 1996; Hernandez-Verdun, 2006; Raska et al., 2006) and, in particular, in plants (Medina et al., 2000; Shaw and Doonan, 2005; Sa´ez-Va´squez and Medina, 2008), always using cellular models characterized by high cell proliferating activity. Furthermore, regulation of ribosome production is controlled by a subset of nucleolar proteins whose activity is, in turn, controlled by factors regulating cell cycle progression (Klein and Grummt, 1999; Sa´ez-Va´squez and Medina, 2008). The major nucleolar protein is nucleolin, a protein conserved in animals, plants and yeast, whose levels are correlated with the rate of functional activity of the nucleolus in exponentially growing cells (Ginisty et al., 1999; Sa´ez-Va´squez and Medina, 2008). The analysis of these parameters in root meristematic cells of samples grown in space and in the corresponding ground controls has led us to define the alterations induced by microgravity on cell growth and proliferation. We found a striking decoupling of these two processes that are strictly coordinated in ground conditions.
Materials and methods The results presented in this paper correspond to an experiment performed on the ISS in the course of the Spanish ‘‘Cervantes’’ Soyuz mission, which took place in October 2003 (van Loon et al., 2007). Samples obtained from this experiment were complemented with some others, coming from the STS-84 ‘‘Atlantis’’ Space Shuttle mission of NASA from May 1997, in which Arabidopsis seedlings were germinated and grown in space in similar conditions (Kiss et al., 1999). In addition, a ground control experiment in a random positioning machine (RPM), a reliable simulator of microgravity (van Loon, 2007b), was performed under the same conditions, and using the same hardware as in the ISS experiment. An overall schematic view of the scenarios used in the different experiments is presented in Fig. 1. ISS experiment Seeds of Arabidopsis thaliana (L.), Heynh., ecotype ‘‘Columbia’’ (Col-0), which had been stored for at least three weeks at 4 1C for vernalization, were sterilized in 1.25% (v/v) sodium hypochlorite
and 1% (v/v) Triton X-100 for 10 min and dried onto a sheet of filter paper (Whatman CHR1), 5 3 cm (approx. 90 seeds per sheet). They were prepared to be sent to space and then to germinate, grow and be fixed in-flight, using a device prepared and assembled according to the ‘‘Berlingot-Ampoule’’ concept, first introduced by the ‘‘Groupement Scientifique en Biologie et Me´decine Spatiales’’ (GSBMS, Toulouse, France) (Tixador et al., 1981). Briefly, two sets of 10 ampoules, 30 mL each, one set containing culture medium (Murashige and Skoog’s, Duchefa) and the other set containing a fixative solution of 8% (v/v) paraformaldehyde (PFA, EM grade, EMS, Fort Washington PA, USA) in PBS, were introduced in a ‘‘Berlingot’’, that is, a double-wall plastic bag, together with seeds and filter paper and then sealed. Two ‘‘Berlingots’’ were then introduced into hermetic Biorack type I/E biocontainers supplied with the device known as motorized ampoule breaker assembly (MAMBA), a technical development of Dutch Space B.V. (Leiden, The Netherlands) in order for the semi-automatic breakage of ampoules. A detailed description of this hardware, including graphic documentation, was published previously (Matı´a et al., 2007). Once in the ISS, the experiment was activated by breaking the first set of ampoules and leaving the culture medium to wet the filter paper. This promoted germination of seeds. At this moment, biocontainers were transferred to an incubator at a constant temperature of 22 (70.5) 1C. After 4 d, the second set of ampoules was broken, releasing the fixative solution, which, mixed with the same amount of culture medium already present within the ‘‘Berlingot’’, gave a paraformaldehyde (PFA) concentration of 4%. All operations were performed semi-automatically, only by activating an electric motor within each biocontainer. Therefore, biocontainers remained hermetically closed throughout all the process, so that seedling germination and growth occurred in darkness and the available air was that initially contained in each Biorack container (the ‘‘Berlingot’’ plastic walls are permeable to air). The experiment materials returned back to the Earth on the same day of sample fixation. The temperature of the biocontainers progressively rose from 22 to 27 1C during the journey (approximately 8 h). Once on Earth, they were stored in an active refrigerated container at 4 1C, where they were transported from the landing site in Kazakhstan to the ‘‘Star City’’ Russian Space Complex, near Moscow (this transport took another period of approximately10 h). In this location, the experimental materials were received by the investigators. Biocontainers were immediately opened, and samples were photographed and transferred to PBS for washing (3 10 min) and partially dehydrated in an ethanol series until 70% ethanol; in this medium they were transported to Madrid, Spain, for final processing for microscopy and immunocytochemistry, including full dehydration and embedding in LR White resin (London Resin Co., UK).
Space shuttle (STS-84 mission) experiment This experiment was carried out in the ESA payload ‘‘Biorack’’, flown in the American Space Shuttle ‘‘Atlantis’’ (Kiss et al., 1999). In this experiment, Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija Ws) seeds germinated and seedlings grew in orbit at 22 1C for 90 h in the darkness. A red light pulse of 10 min was given 24 h after seed soaking to stimulate germination. Fixation was initiated in-flight with 4% (v/v) glutaraldehyde in 100 mmol/L phosphate buffer and extended for 5.5 d, until samples were recovered on ground, where processing continued with washing in buffer, dehydration in ethanol series and embedding in Quetol 651 resin (Guisinger and Kiss, 1999; Kiss et al., 1999). A control experiment in-flight, using a 1g centrifuge, was performed under the same conditions.
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Random positioning machine (RPM) experiment This experiment was performed in the RPM located in the Dutch Experiment Support Center (DESC), Vrije Universiteit, Amsterdam, The Netherlands. This machine does not remove gravity, but its action becomes omnilateral instead of unilateral, resulting in the effect that plants are not capable of responding to a definite gravity vector (van Loon, 2007b). The biological material, hardware used, times and temperatures of growth and sample processing were an exact replica of the ISS experiment. The rotational velocity of the RPM frames was randomized with a maximum of 7601 s 1; the interval was set at random. The samples were positioned in the center of the inner frame, and the largest radius was, at maximum, 5 cm to the outermost biocontainer. This resulted in a maximum residual gravity of less than 10 4g (van Loon, 2007b).
Control 1g experiments Ground 1g control experiments were performed in our laboratory for the three experiments described above, namely that of the ISS, that of the Space Shuttle and that of the RPM. In these control experiments the biological material, hardware used, times and temperatures of growth and sample processing were the same or exact replicas as in the reference experiments. In the case of the RPM, control biocontainers were positioned in the outer static frames of the instrument.
Microscopy, immunocytochemistry and quantification From the materials embedded in resin, semithin sections, 2 mm thick, were cut and observed unstained under a Leica DM2500 microscope equipped with phase contrast. Images were digitally recorded with a Leica DFC320 CCD camera. For ultrastructural studies, ultrathin sections were mounted on Formvar-coated nickel grids stained with uranyl acetate and lead citrate and observed in a Jeol 1230 electron microscope, operating at 100 kV. For immunogold labeling, ultrathin sections were incubated with an anti-nucleolin antibody (kindly supplied by Dr. J. Sa´ez-Va´squez, CNRS, Perpignan, France) diluted 1:100, for 90 min at room temperature (RT), washed repeatedly and incubated with goat anti-rabbit IgG secondary antibody coupled with 10 nm colloidal gold particles (Sigma-Aldrich, St Louis MO, USA), diluted 1:50, for
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1 h, at RT. Prior to observation, grids were counterstained with uranyl acetate, either alone or followed by lead citrate. Quantitative measurements (seedling and root length, number of cells per mm in root cell files, cross-sectional area of nucleoli, proportion of nucleolar granular component (GC) and density of gold particles) were carried out on digital images using the quantitation software ‘‘QWin Standard’’ (Leica Microsystems). Measurements of cell parameters were performed in a zone of 50 730 mm above the quiescent center (approx. 100730 mm from the root tip), corresponding to the highest rate of cell division, according to the available literature (Beemster and Baskin, 1998). In this zone, epidermal, cortical and endodermal cells were selected. The number of samples measured was: 20 seedlings for seedling length, 10 roots for number of cells per mm, 60 nucleoli (light microscopy) for cross-sectional area and 15 nucleoli (electron microscopy) for GC proportion and density of gold particles. Statistical analysis of data was performed using t SPSS 13.0 software. Description of quantitative variables was performed using means and standard deviations after checking normality with the Kolmogorov–Smirnov test. Mean values were compared using the Student’s t-test for independent samples; differences were considered significant for a bilateral a value lower than 0.05.
Results Seeds germinated in either real or simulated microgravity showed a high rate of germination (more than 75%), comparable with the rate obtained in ground 1g control conditions. In all cases, germination and growth for 4 d in darkness produced etiolated seedlings, lacking any green part and showing a highly developed hypocotyl (Fig. 2a). As expected, growth orientation of roots and hypocotyls in seedlings from both real and simulated microgravity experiments was random, whereas seedlings from 1g controls appeared oriented with respect to the gravity vector. Seedlings grown in space and grown in the random positioning machine (RPM) showed a longer size than those of the ground 1g control, and the differences were significant (Fig. 2). However, no significant differences in length were found between the flight samples and the RPM samples (Fig. 2b). The increase in length of the seedlings grown in either real or simulated microgravity was due to an increased length of both the root and the hypocotyl (Fig. 2a). Root length was significantly longer in the flight samples and the RPM samples, but the difference with respect to the
Fig. 2. a: Seedlings of Arabidopsis thaliana grown in space (flight, left), in simulated microgravity in the Random Positioning Machine (RPM, center) and in control 1g ground conditions (right). Growth in darkness produced etiolated seedlings with a very developed hypocotyl. Compared at the same magnification, a longer size of seedlings grown in microgravity (either real or simulated) stands out clearly. b, c: Quantitative measurement of the seedling length (b) and the root length (c) from the spaceflight experiment and its associated ground control experiments. Mean and standard deviation values are graphically represented. Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (p o 0.05). N = 20 seedlings for each condition.
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Fig. 3. Semithin (2 mm thick) sections of the root distal part from samples grown under the different conditions, observed under the phase-contrast microscope. The difference in shape and size between the larger and more regular cells of the cortical cylinder (CoC) with respect to the longer and narrower cells of the central cylinder (CeC) is clearly appreciated. Cells of the cortical cylinder, organized as the files called epidermis (Ep), cortex (Co) and endodermis (En), constitute the ground tissue root meristem. There are more of these cells in each file, and they are smaller, in the microgravity-grown samples (flight and RPM) than in the ground 1g control. QC: Quiescent center. Col: Columella. LRC: Lateral root cap.
Fig. 4. Estimation of the cell proliferation rate in the cortical root meristematic cell files, by counting the number of cells per millimeter in each file. Ten roots (root meristems) were used for each condition. Mean and standard deviation values are graphically represented. Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (p o0.05).
ground 1g control was less than the difference recorded for whole seedlings (Fig. 2c). Semithin sections of the root meristem obtained from all samples, observed under phase-contrast microscopy, showed the files of cells corresponding to both the central and the cortical cylinder. At the root tip, quiescent center and columella cells were visible (Fig. 3). In the three files of the cortical cylinder (epidermis,
cortex and endodermis), and in a zone of 50 730 mm above the quiescent center (approx. 100 730 mm from the root tip) we counted the number of cells per mm. This number was significantly higher in the flight and the RPM samples compared to the ground 1g control; no significant difference was found between the flight and the RPM samples (Fig. 4). Consequently, samples grown in either real or simulated microgravity showed a higher cell proliferation rate (notice that, in addition to containing more cells per mm, roots were longer in these conditions; see Fig. 2) and the cells were shorter than in control conditions. In addition to the quantitative demonstration, this feature was clearly observed in microscopic images (Fig. 3). Estimations of the alterations in cell growth induced by microgravity were based on certain morphological and morphometrical features of the nucleolus that have been demonstrated to be functionally associated with the rate of ribosome biogenesis in proliferating cells. The features are the nucleolar size, the relative proportion and distribution of nucleolar subcomponents and the levels of the nucleolar protein nucleolin. To investigate these features we used samples fixed in glutaraldehyde, as well as those fixed in PFA, together with their respective ground 1g controls. Fixation in glutaraldehyde was necessary for clear discrimination of nucleolar subcomponents; fixation in PFA was necessary for immunogold localization of nucleolin. Nevertheless, comparison of the overall nucleolar ultrastructure in the two sets of experiments showed that, for the purpose of our studies, samples were comparable (compare Fig. 5a–c with Fig. 5d–f and Fig. 7). In samples fixed with glutaraldehyde, nucleolar subcomponents were clearly discernible (Fig. 5a–c). In all cases, the nucleolar ultrastructure corresponded to that of a proliferating cell actively engaged in the production of ribosomes, showing
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Fig. 5. a–c: Ultrastructure of nucleoli from root meristematic cortical cells fixed in glutaraldehyde in space and its associated ground control experiment. a: Sample grown in space (flight lg); b: sample grown in the 1g centrifuge, on board of the spacecraft (flight 1g), and c: control sample, grown on Earth (ground 1g). Notice the difference in size between the three samples. The three basic nucleolar subcomponents are clearly distinguished, namely fibrillar centers (arrows), dense fibrillar component (DFC) and granular component (GC). Fibrillar centers are larger and less numerous in the flight lg sample, whereas granular component is more abundant in both samples grown at 1g, and this component appears interspersed with territories of dense fibrillar component. d–f: Ultrastructure of nucleoli from root meristematic cortical cells fixed in paraformaldehyde in space and its associated ground control experiments. d: sample grown in space (flight); e: sample grown in the random positioning machine (RPM), under simulated microgravity, and f: control sample, grown on Earth (ground 1g). Notice the difference in size between the three samples. Discrimination of nucleolar subcomponents is more difficult than with glutaraldehyde fixation. However, fibrillar centers are clearly distinguished (arrows) and the granular component (GC) can be differentiated from the dense fibrillar component (DFC), mostly in the peripheral zones. The pattern of number and size of fibrillar centers is similar to that described for glutaraldehyde-fixed samples. Scale bars indicate 1 lm.
features such as an abundant GC (which accounts for approx. 50% of the nucleolar volume) and small FCs of the homogeneous type. However, the nucleolar size was significantly smaller in samples grown in space under microgravity than in samples grown in the 1g centrifuge in space and in the ground 1g control. The nucleolar size in these two latter samples did not show significant differences (Fig. 6a). Considering the amount and distribution of nucleolar components, the microgravity-grown samples showed the GC occupying a peripheral position with respect to the dense fibrillar component (DFC) and FCs, which occupied a central position in the section of the nucleolus (Fig. 5a). In contrast, in the 1g samples (either centrifuge or ground) the GC appeared intermingled with masses of the DFC, which were observed interspersed in the nucleolar section, with small FCs in their interior (Fig. 5b–c). Quantitatively, the GC accounted for 54–57% of the nucleolar volume in 1g samples, whereas it was only around
45% of nucleoli of cells from samples grown in microgravity (Fig. 6a). Taking into account the difference in size between nucleoli from the two types of samples, the amount of nucleolar GC was severely depleted by growth in spaceflight under microgravity relative to the 1g gravity conditions. In fact, practically all of the differences in size between nucleoli from cells grown in various gravity conditions were accounted for by the GC, with the fibrillar components (DFC and FCs) showing a minimal contribution (Fig. 6a). The results of PFA fixation were in agreement with those obtained with glutaraldehyde fixation. The differences were a moderate shrinkage of the organelle, which was a consequence of the reduction in the quality of fixation (Lerı´a et al., 2004), which affected equally to all the samples, whatever the growth conditions, and, as previously mentioned, a difficult discrimination of subnucleolar components, mostly the GC from the DFC,
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Fig. 6. Nucleolar size, measured as cross-sectional area, in root meristematic cortical cells fixed in glutaraldehyde (a) and in paraformaldehyde (b) in space and its associated control experiments. Absolute values are lower for paraformaldehyde- than those recorded for glutaraldehyde-fixed samples, due to poorer quality of fixation. The reduction affected equally to all conditions. Mean and standard deviation values are graphically represented. Dotted areas in the bars indicate the proportion occupied by granular component (GC). Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (p o 0.05). N = 60 nucleoli for each condition, for nucleolar area; N =15 nucleoli for each condition, for GC proportion.
Fig. 7. Immunogold electron microscopic localization of the nucleolar protein nucleolin in root meristematic cortical cells fixed in paraformaldehyde in space, and its associated ground control experiments. From left to right, sample grown in space (flight), sample grown in the random positioning machine (RPM), under simulated microgravity, and control sample, grown on Earth (ground 1g). In general, gold particles appear in the nucleolus, preferentially located in the dense fibrillar component (DFC) surrounding fibrillar centers (arrows). This localization is more conspicuous in the ground 1g control, whereas in samples grown in microgravity (real or simulated), gold particles are more dispersed throughout the nucleolar area. Fibrillar centers themselves, as well as the granular component (GC) appear devoid of labeling. Scale bars indicate 1 mm.
and mainly in the boundary zones (Fig. 5d–f and Fig. 7). In this case, the nucleoli were significantly smaller in the flight and the RPM samples (i.e. those grown under microgravity, either real or simulated) relative to the ground 1g control (Fig. 6b). For subnucleolar components, FCs appeared smaller and more numerous in the ground 1g control (Fig. 5f). Finally, the quantitative immunolocalization of the major nucleolar protein, nucleolin, was used as an additional indicator of the rate of ribosome biogenesis. Electron microscopic immunolocalization is advantageous since it not only allows the
estimation of the levels of the protein, but also its intranucleolar distribution, which is a feature with functional significance (Pontvianne et al., 2007). Nucleolin in all cases was located in the DFC of the nucleolus, near FCs, where it was previously described to be located in active nucleoli of plant meristematic cells. This localization was more evident in the ground 1g control than in the flight and RPM samples, in which the gold particles appeared to be more dispersed throughout the nucleolar area, resembling the pattern observed in low-active nucleoli (Fig. 7). Quantitatively, ground 1g control cell nucleoli showed a
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Fig. 8. Quantitative measurement of the nucleolin levels, estimated by the density of gold particles (no. of gold particles per mm2), after immunolabeling, in root meristematic cortical cells from the spaceflight experiment and its associated ground control experiments. Mean and standard deviation values are graphically represented. Asterisks indicate a statistically significant difference from the ground 1g control, according to the Student’s t-test (po 0.05). N = 15 nucleoli for each condition.
significantly higher density of labeling than nucleoli from either flight or RPM-grown samples, which did not show significant differences between them (Fig. 8). This was an additional confirmation of the depletion of the rate of ribosome biogenesis occurring in cells grown in microgravity, either real (spaceflight) or simulated (RPM). Remarkably, for all measurements performed, no significant differences were found between samples grown in spaceflight and in the RPM. Measurements performed in the ground 1g control samples and in samples grown in the 1g centrifuge during spaceflight were also not significantly different.
Discussion The spaceflight environment comprises a collection of conditions, such as microgravity, radiation and other factors that can influence life processes. The differentiation of microgravity effects from the other environmental factors that are present in space is the main reason to apply an in-flight 1g centrifuge (providing all space factors but microgravity) and to perform ground control experiments using devices that simulate microgravity, such as RPM (providing microgravity, but no other space factors), whenever possible (van Loon, 2007a). According to the results obtained in this study and comparing the measured parameters in the different environmental conditions tested, we conclude that microgravity is the most important source of alteration. This conclusion comes from the comparison of the results obtained in the spaceflight experiment (real microgravity) with those obtained in the RPM (simulated microgravity), which did differ significantly. The conclusion is also supported by the comparison of the results obtained from the flight 1g experiment and the ground 1g experiment, which were statistically similar. Additionally, RPM was confirmed in our work as a good and reliable simulator of microgravity, in line with previous published results using other parameters, such as the position of amyloplasts in columella cells, or the changes in shape of attached cells in culture (Kraft et al., 2000; van Loon, 2007b). The enormous advances in recent years producing knowledge of genomic data, in particular in the model plant species A. thaliana, has
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led to the understanding that a sizeable number of genes modify their expression pattern in response to changes in the environmental gravity conditions (Moseyko et al., 2002; Centis-Aubay et al., 2003; Kimbrough et al., 2004; Martzivanou et al., 2006; Salmi and Roux, 2008). In some cases, the gravity alteration consisted of the reorientation of the growth direction, sometimes accompanied by a simple mechanical stimulation (Moseyko et al., 2002; Kimbrough et al., 2004). In other cases, ground-based microgravity simulators (clinostat or RPM) were used to induce this alteration (Centis-Aubay et al., 2003; Martzivanou et al., 2006), and in the last case, the analysis was performed on fern spores germinated in spaceflight (Salmi and Roux, 2008). The identification of altered genes reveals a heterogeneous group comprising transcription factors, modulators of cell wall features, elements of early signaling cascades and signal transduction pathways, hormone-responsive elements and other genes of unknown function. Several of these genes also appear altered in plants affected by known environmental stresses (dehydration, cold, pathogen response). Some general conclusions have been reached from these genomic studies, such as the identification of the gravity alteration as an actual stress condition for the plant, the existence of a mechanism of signaling, and a signal transduction cascade as a response to the alteration and the involvement of hormones, and specifically auxin in this response. However, these types of analysis have supplied few data on the cellular mechanisms altered by the gravity modification. In particular, changes in basic cellular processes influencing plant growth and development are scarcely documented in these studies, even though some of the genes modified in their expression may influence these cellular processes. Therefore, the genomic approach, while of great importance and interest, should be supplemented by other experimental approaches providing complementary information by means of the use of additional parameters, in order to get the full picture of the functional alterations induced by the environmental change. Our results clearly show that growth in the microgravity environment of spaceflight produces substantial alterations in root meristematic cell growth and proliferation. Cell proliferation appears enhanced, whereas the cell size and the rate of production of ribosomes (both taken as indicators of cell growth) appear depleted. With respect to ribosome biogenesis, all morphological and morphometrical features of nucleoli, together with the immunocytochemical estimation of the levels of the nucleolar protein nucleolin, indicated a depletion in the rate of pre-rRNA transcription and processing, according to the morpho-functional models, previously published and generally agreed upon (Shaw and Jordan, 1995; Medina et al., 2000). A lowering in the rate of ribosome biogenesis was previously detected in our laboratory in root meristematic cells from clinorotated samples (Sobol et al., 2005; 2006), but it was not put in relation to other cellular processes. Cell proliferation and cell growth, which are closely associated and interdependent processes in this cell type under ground gravity conditions, appear divergent in the microgravity environment. Our interpretation of this divergence is that the cell cycle is altered by the change in gravity conditions. It is known that the major part of ribosome production (and consequently, of cell growth) in cycling cells occurs in G2 as a preparation for mitosis (Sa´ez-Va´squez and Medina, 2008). A shortening of the G2 phase could result in a reduction in cell growth/ribosome biogenesis and in an acceleration of cell division, producing a greater number of shorter cells, i.e. the experimental result we obtained in the spaceflight and RPM tests. Checkpoints for cell size occur indeed just in the G2 stage and in the G2-mitosis transition (Mizukami, 2001; Sugimoto-Shirasu and Roberts, 2003; Inze´ and De Veylder, 2006). Changes in cell cycle duration have been described in young seedlings grown in spaceflight (Perbal and Driss-E´cole, 1994; Yu et al., 1999). In these experiments, carried out in the Spacelab IML-1
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and IML-2 missions, lentil seedlings were grown for 28–29 h, i.e. the time necessary to complete the first cell cycle after germination. The percentages of the different cell cycle phases (G1, S, G2, M) were determined after Feulgen staining. In the two experiments, samples grown in space were enriched in earlier phases with respect to 1g controls, and the authors concluded that the cell cycle was slowed down in microgravity. However, this conclusion does not take into account a possible different effect of microgravity depending on the time of exposure and/or on the phase of development of the plant or seedling (Claassen and Spooner, 1994; Aarrouf et al., 1999). In fact, this time of growth under microgravity conditions was quite different in these experiments (28–29 h) and in the experiment reported in the present paper (4 d). The origin of the observed alterations in cell proliferation and cell growth in a weightless environment could be either the transduction of the mechano-signal sensed by columella cells and responsible for gravitropism, or the perception of independent signals by cells not apparently specialized in gravity sensing (Kordyum, 1997). In favor of the first possibility is the fact that the transduction of the gravitropic signal finally results in the change of the distribution of auxin streams in the root, producing lateral gradients of this hormone (Boonsirichai et al., 2002). It is known that auxin plays a role in the regulation of cell growth and cell proliferation by molecular mechanisms that are beginning to be understood (Himanen et al., 2002; David et al., 2007). Studies carried out on cultured animal cells in weightlessness (which obviously lack mechano-receptors comparable to those of statocytes), showing alterations in lymphocyte proliferation (Cogoli and Cogoli-Greuter, 1997), melanoma growth and tumorigenicity (Dai et al., 2007), or osteogenesis of bone marrow stem cells (Taga et al., 2006), among others, would support the second alternative. The alteration of basic and essential cellular processes in the root meristem as a consequence of gravity changes indicate that this environmental modification can be considered as a real stress for the plant that may have consequences in plant morphogenesis and development. Furthermore, it could be hypothesized that plant responses described for other abiotic stress conditions, such as the so-called ‘‘stress-induced morphogenic response’’ (SIMR) (Potters et al., 2007), could also be found as a consequence of the growth in a microgravity environment. Certainly, the alterations that we have observed do not fit exactly with the model of SIMR, but similar parameters (cell growth, cell proliferation and seedling morphology) are affected and the response observed after the gravitational stress is indeed a morphogenic response. Interestingly, the cellular and molecular mechanisms associated with SIMR and triggering its effects are auxin transport and/or metabolism, cell cycle regulation and production of reactive oxygen species (ROS), a class of molecules with high oxidative capacity (Potters et al., 2007). In relation to these molecules, a group of genes belonging to the functional category of the oxidative burst, related to the plant defense mechanisms, was found to be overexpressed after gravitational stimulation (Kimbrough et al., 2004). It would be interesting to analyze the presence of ROS (and their possible induction) in plants grown under microgravity or altered gravity conditions. In conclusion, microgravity conditions during spaceflight produce stress for seed germination and seedling growth which, at the cellular level, results in the divergence of cell proliferation and cell growth in root meristematic cells. These two processes are strictly related to each other in this cellular type in normal ground gravity conditions. Even though the technical limitations of the spaceflight experiment did not allow us to perform additional experiments, we can hypothesize that shortening of one or more phases of the cell cycle, produced by the alteration of at least some checkpoints responsible for their regulation, could
account for the observed effects. Experiments involving the analysis of the expression of key factors in the regulation of cell cycle progression at the level of these checkpoints, using more points of seedling development for data recording, are currently in progress (Matı´a et al., 2009) and should contribute to clarification of this important problem.
Acknowledgements The authors thank the European Space Agency (ESA) astronaut Pedro Duque (ISS experiments) and the astronauts of the STS-84 Mission (Space Shuttle experiments) who performed the in-flight operations of the experiments reported in this paper. We also thank Mrs. Mercedes Carnota (CIB-CSIC, Madrid, Spain) and Mrs. Brigitte Eche (GSBMS, Toulouse, France) for excellent technical assistance in the ground experiments. The generous gift of anti-nucleolin antibody by Dr. Julio Sa´ez-Va´squez (CNRS, Perpignan, France) is gratefully acknowledged, as well as the supply of MAMBAs by Dutch Space B.V. (Leiden, The Netherlands). The ESA personnel at ESTEC (Noordwijk, The Netherlands) were of great help in the preparation of the ISS Mission; in particular, we would like to thank Mr. Jesu´s Jime´nez, Ms. Natalie Pottier and Mr. Fabrizio Festa, as well as Dr. Didier Chaput (CNES, Toulouse, France). This work was supported by grants from the Spanish ‘‘Plan Nacional de I+D+i’’, Refs. nos. ESP2001-4522-PE and ESP2006-13600-C02-02, and the use of RPM, by Grant no. MG-057 (SRON, NL). References Aarrouf J, Darvelley N, Demandre C, Razafindranboa N, Perbal G. Effect of horizontal clinorotation on the root system development and on lipid breakdown in Rapeseed (Brassica napus) seedlings. Plant Cell Physiol 1999;40:396–405. Barmicheva EM, Grif VG, Tairbekov MG. Growth and structure of cells in the maize root apex under space flight conditions. Cytology 1989;31:1324–8. Baserga R. Is cell size important?. Cell Cycle 2007;6:814–6. Beemster GTS, Baskin TI. Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol 1998;116:1515–26. Boonsirichai K, Guan C, Chen R, Masson PH. Root gravitropism: an experimental tool to investigate basic cellular and molecular processes underlying mechanosensing and signal transmission in plants. Annu Rev Plant Biol 2002;53: 421–447. Campbell WF, Salisbury FB, Bugbee B, Klassen S, Naegle E, Strickland DT, et al. Comparative floral development of Mir-grown and ethylene-treated, earthgrown super dwarf wheat. J Plant Physiol 2001;158:1051–60. Centis-Aubay S, Gasset G, Mazars C, Ranjeva R, Graziana A. Changes in gravitational forces induce modifications of gene expression in A. thaliana seedlings. Planta 2003;218:179–85. Claassen DE, Spooner BS. Impact of altered gravity on aspects of cell biology. Int Rev Cytol 1994;156:301–73. Cogoli A, Cogoli-Greuter M. Activation and proliferation of lymphocytes and other mammalian cells in microgravity. Adv Space Biol Med 1997;6:33–79. Cowles JR, Scheld HW, Lemay R, Peterson C. Growth and lignification in seedlings exposed to eight days of microgravity. Ann Bot 1984;54:33–48. Dai ZQ, Wang R, Ling SK, Wan YM, Li YH. Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Proliferation 2007;40:671–84. Darbelley N, Driss-E´cole D, Perbal G. Elongation and mitotic activity of cortical cells in lentil roots grown in microgravity. Plant Physiol Biochem 1989;27: 341–347. David KM, Couch D, Braun N, Brown S, Grosclaude J, Perrot-Rechenmann C. The auxin-binding protein 1 is essential for the control of cell cycle. Plant J 2007;50:197–206. Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, et al. Cellular organisation of the Arabidopsis thaliana root. Development 1993;119:71–84. Driss-E´cole D, Schoevaert D, Noin M, Perbal G. Densitometric analysis of nuclear DNA content in lentil roots grown in space. Biol Cell 1994;81:59–64. Feldman LJ. Root gravitropism. Physiol Plant 1985;65:341–4. Ferl R, Wheeler R, Levine HG, Paul AL. Plants in space. Curr Opin Plant Biol 2002;5:258–63. Fujie M, Kuroiwa H, Suzuki T, Kawano S, Kuroiwa T. Organelle DNA Synthesis in the quiescent centre of Arabidopsis thaliana (Col.). J Exp Bot 1994;44:689–93. Ginisty H, Sicard H, Roger B, Bouvet P. Structure and functions of nucleolin. J Cell Sci 1999;112:761–72.
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