Short running title: Plant cell proliferation and growth in spaceflight

Corresponding author : Dr. F. Javier Medina Centro de Investigaciones Biológicas (CSIC) Ramiro de Maeztu 9 E-28040 Madrid, Spain Phone: +34 91 8373112. Fax: +34 91 5360432. Email: fjmedina@cib.csic.es

Plant cell proliferation and growth are altered by microgravity conditions in spaceflight§

Isabel Matíaa, Fernando González-Camachoa, Raúl Herranza, John Z. Kissb, Gilbert Gassetc, Jack J.W.A. van Loond, Roberto Marcoe and Francisco Javier Medinaa*

aCentro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain bDepartment of Botany, Miami University, Oxford OH 45056, USA cGSBMS, Université “Paul Sabatier”, Faculté de Médecine de Rangueil, Bât A2, 133 route de Narbonne, F-31062 Toulouse, France dDutch Experiment Support Center (DESC), OCB-ACTA, Vrije Universiteit, Amsterdam, The Netherlands eDepartamento de Bioquímica-I.I. Biomédicas “Alberto Sols” (UAM-CSIC), Arzobispo Morcillo 4, E-28029 Madrid, Spain

*Corresponding author.

§Our colleague and friend Professor Roberto Marco passed away June 27th, 2008. This paper is affectionately dedicated to his memory.

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Summary

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4 days

and then they were fixed in flight with paraformaldehyde. The experiment was replicated on ground in a

Random Positioning Machine, an effective simulator of microgravity. Furthermore, samples from a different

space experiment, processed in a similar way, but fixed in glutaraldehyde, including a control flight

experiment in a 1 g centrifuge, were also used. In all cases, comparisons were done with ground controls at 1

g. Seedlings grown in microgravity were significantly longer than the ground 1 g controls. The cortical root

meristematic cells were analyzed for investigating 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

it was found to be higher in microgravity-grown samples than in the control 1 g. 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 1 g. 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.

Keywords: cell cycle; microgravity; nucleolus; ribosome biogenesis; root meristem.

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. MAMBA, Motorized Ampoule Breaker Assembly. NASA:

National Aeronautics and Space Administration. PBS, Phosphate-Buffered Saline. PFA, Paraformaldehyde.

ROS, Reactive Oxygen Species. RPM, Random Positioning Machine. RT, Room Temperature. SIMR, Stress

Induced Morphogenic Response.

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Introduction

Gravity plays a role in plant development and shaping and its alteration induces changes in these

processes (Perbal, 2001; Ferl et al., 2002). Investigating this topic is necessary for the current programs of

space exploration, in which the design and building of successful life support systems will be mandatory in

long-duration spaceflights. .

The influence of gravity is easily detected in plants since 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 a key role in plant growth and development (Teale et al., 2006).

Most studies on the effects of gravity alteration on plant growth and development were focused on

the root. The length of the primary root in microgravity, compared with 1 g ground control, was found 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). Among the causes for these discrepancies, it could

be mentioned 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; Campbell et al., 2001). In addition, it was 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 on real microgravity.

The processes of growth, differentiation and development affect the whole plant, but they rely on

cellular mechanisms, namely 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 the 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 is capable of modulating the activity of

meristematic cells. Results obtained in space experiments showed that the progression of cell cycle is

modified in lentil seedlings grown for 30 h in microgravity (Legué 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 S phase and an increased proportion of cells in G1 phase,

suggesting that G1/S transition is modulated by gravity. Mitotic index was shown to be depleted in plants

grown in space (Barmicheva et al., 1989; Driss-École et al., 1994); however, this result does not necessarily

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mean a depletion of the proliferation rate, but it can 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 the ground 1 g 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-École 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 days and chemical fixation of samples, accompanied by the

corresponding controls. All the postflight analyses have been 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, so that their only functional program

is to grow and divide continuously, 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 µm from

the quiescent center (Beemster and Baskin, 1998).

In this zone of the root tip, we have analyzed a series of cellular parameters which are highly reliable

indicators of the status of proliferation and growth of cells in order to assess the variations in these functions,

originated by a change in gravity conditions. Regarding proliferation, we have 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 largely determined by the activity of

RNA polymerase I that controls ribosomal RNA synthesis and ribosome biogenesis (Baserga, 2007).

Ribosome biogenesis occurs in a well-defined nuclear territory – the nucleolus, and it has been largely and

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 and the

granular component. 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; Sáez-Vásquez and Medina, 2008), always

using cellular models characterized by a 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; Sáez-Vásquez and Medina, 2008). The major

nucleolar protein is nucleolin, a protein conserved in animals, plants and yeast, whose levels are correlated

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with the rate of functional activity of the nucleolus in exponentially growing cells (Ginisty et al., 1999; Sáez-

Vásquez 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 have found a striking decoupling of these two processes, which 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. 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 in 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 4ºC 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 Médecine Spatiales” (GSBMS-CNES, Toulouse,

France) (Tixador et al., 1981). Briefly, two sets of 10 ampoules, 30 L 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 MAMBA (Motorized AMpoule Breaker Assembly), 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 (Matía 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 (±0.5) ºC. After 4 days, the second set of

ampoules was broken, releasing the fixative solution, which, mixed with the same amount of culture medium

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already present within the “Berlingot”, gave a 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 27ºC during the journey (approximately 8 h).

Once on Earth, they were stored in an active refrigerated container at 4ºC, 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 10 h, approx.). In this location, the experiment materials were received by the investigators.

Immediately, biocontainers were opened, 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

22ºC, for 90 h, in the darkness. A red light pulse of 10 min was given 24 hrs after seed soaking, to stimulate

germination. Fixation was initiated in flight with 4% (v/v) glutaraldehyde in 100 mol/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 1 g centrifuge, was performed in the same conditions.

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 ± 60°·

s1; 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 104 g (van Loon, 2007b).

Control 1 g experiments —Ground 1 g 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.

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Microscopy, Immunocytochemistry and Quantification —From the materials embedded in resin, semithin

sections, 2 m 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. Sáez-Vásquez,

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 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 ± 30 µm above the

quiescent center (approx. 100 ± 30 µm 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, respectively. Statistical analysis of

data was performed using the software “SPSS 13.0”. The description of quantitative variables was done

using mean and standard deviation, after checking normality with the Kolmogorov-Smirnov test. Mean

values were compared by the Student’s t test for independent samples; differences were considered

significant for a bilateral 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 days 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 at random, whereas seedlings from 1 g controls

appeared oriented with respect to the gravity vector.

Seedlings grown in space, and also those grown in the RPM showed a longer size than those of the

ground 1 g control, with significant differences (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 root and hypocotyl

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(Fig. 2a). Root length was significantly longer in the flight samples and the RPM samples, but the difference

with respect to the ground 1 g 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

microscope, 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 ± 30 µm above the quiescent center (approx. 100 ±

30 µm 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 1 g 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.

Besides the quantitative demonstration, this feature could be clearly observed in microscopical images (Fig.

3).

Estimations of the alterations in cell growth induced by microgravity were based on certain

morphological and morphometrical features of the nucleolus which have been demonstrated to be

functionally associated with the rate of ribosome biogenesis in proliferating cells. These 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 1 g controls. Fixation in glutaraldehyde was

necessary for a good 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

Figs. 5a-c with Figs. 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 features such as an abundant granular component (GC) (which accounts

for approx. 50% of the nucleolar volume) and small fibrillar centers (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 1 g centrifuge in space and in the ground 1 g 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 1 g 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 1 g

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

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nucleolar GC was severely depleted by growth in spaceflight, under microgravity, regarding the 1 g gravity

conditions. In fact, practically all the difference in size between nucleoli from cells grown in various gravity

conditions was accounted for by the GC, the fibrillar components (DFC and FCs) having a minimal

contribution (Fig. 6a).

The results of PFA fixation were completely 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 (Lería 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, and mainly in the boundary zones (Figs. 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) with respect to the ground 1 g control (Fig. 6b). Regarding subnucleolar

components, FCs appeared smaller and more numerous in the ground 1 g 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 microscopical 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 appeared in

all cases 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 1 g control than

in the flight and RPM samples, in which the gold particles appeared to be more dispersed throughout the

nucleolar area, somehow resembling the pattern observed in low-active nucleoli (Fig. 7). Quantitatively,

ground 1 g control cell nucleoli showed a 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 have been found between

samples grown in spaceflight and in the RPM. On their part, measurements performed in the ground 1 g

control samples and in samples grown in the 1 g 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 which are present in space is the main reason why to apply an in-flight 1 g 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 work and comparing the measured parameters in

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the different environmental conditions tested, we can conclude that microgravity is the most important

source of alterations. 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 not show

significant differences. The conclusion is also supported by the comparison of the results obtained from the

flight 1 g experiment and the ground 1 g 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 advance in recent years of the knowledge of genomic data, in particular in the model

plant species Arabidopsis thaliana, including technical progress, has 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, accompanied or not 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 space flight (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 appear also altered in plants affected by known

environmental stresses (dehydration, cold, pathogen response). Some general conclusions were 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, specifically auxin in this response. However, this kind of

analysis has not supplied many data on the cellular mechanisms which are 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 which appear modified in their

expression may influence these cellular processes. Therefore, the genomic approach, whose high importance

and interest are not questioned at all, 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 have clearly shown 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. Regarding 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,

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previously published and generally agreed (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 (Sáez-Vásquez 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 have 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; Inzé and De Veylder, 2006).

Changes in cell cycle duration have been described in young seedlings grown in spaceflight (Perbal

and Driss-École, 1994; Yu et al., 1999). In these experiments, carried out in the Spacelab IML-1 and IML-2

missions, lentil seedlings were grown for 28-29 hrs, 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, the result was that samples grown in space were enriched in earlier

phases with respect to 1 g controls, so that 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 hrs) and in the experiment reported in the present paper (4 days).

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). Otherwise, 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,

which may have consequences in plant morphogenesis and development. Furthermore, it could be

11

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 a remarkable 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 (Matía et al., 2009) and should contribute

to clarify this important problem.

Acknowledgements

The authors thank the 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-CNES, Toulouse, France) for excellent technical assistance in the ground experiments. The

generous gift of anti-nucleolin antibody by Dr. Julio Sáez-Vásquez (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. Jesús Jiménez, 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).

12

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Legends of Figures

Figure 1. Schematic representation of the scenarios used in the two space experiments reported in this paper

and their corresponding controls. Note that the timeline is not scaled for the sake of clarity of the different

steps. 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 (22ºC) 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.

Figure 2. a: Seedlings of Arabidopsis thaliana grown in space (Flight, left), in simulated microgravity in the

Random Positioning Machine (RPM, center) and in control 1 g 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 with the ground 1 g control,

according to the Student’s t test (p < 0.05). N= 20 seedlings for each condition.

Figure 3. Semithin (2 m 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 1 g control. QC: Quiescent center. Col: Columella. LRC: Lateral root cap.

Figure 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 with the ground 1 g control, according to the Student’s t test (p < 0.05).

Figure 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 g); b: sample grown

16

in the 1 g centrifuge, on board of the spacecraft (Flight 1 g), and c: control sample, grown on Earth (Ground

1 g). 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 g sample, whereas granular

component is more abundant in both samples grown at 1 g, 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 1 g). 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 m.

Figure 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 with the ground 1 g control, according to the

Student’s t test (p < 0.05). N= 60 nucleoli for each condition, for nucleolar area; N=15 nucleoli for each

condition, for GC proportion.

Figure 7. Immunogold electron microscopical 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 1 g). 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 1 g 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 m.

Figure 8. Quantitative measurement of the nucleolin levels, estimated by the density of gold particles (No. of

gold particles per m2), after immunolabeling, in root meristematic cortical cells from the spaceflight

experiment and its associated ground control experiments. Mean and standard deviation values are

17

graphically represented. Asterisks indicate a statistically significant difference with the ground 1 g control,

according to the Student’s t test (p < 0.05). N= 15 nucleoli for each condition.

Fig. 1

Fig. 2Fig. 2

Fig. 3

Fig 4Fig. 4

Fig. 5

Fig 6Fig. 6

Fig. 7g

Fig. 8