The European Modular Cultivation System (EMCS) is an ESA facility dedicated mainly to Plant Biology experiments but which can also accommodate small animals.
EMCS has been launched to the International Space Station (ISS) in July 2006 and is currently accommodated inside the NASA "Destiny" laboratory module. It is planned to transfer EMCS from "Destiny" to the ESA "Columbus" module in the course of 2008.

EMCS includes a temperature controlled incubator, with two rotors for up to 8 Experiments Containers (ECs), each rotor can be independently set to various g-levels ranging from 1.10^-3 g to 2g. The rotor may also be used as static "0-g"racks, when not rotating. The facility provides automatic gas composition control (including an ethylene removing system) as well as automatic water supply and humidity control at EC level. EMCS is also fitted with an illumination and video-observation system, which allows visible as well as Infra-Red (IR) "dark" on-rotor observation of samples.

Three experiments have been already performed with EMCS: the first one from NASA (TROPI) and the first two ESA experiments: GRAVI-1 (a precursor experiment for GRAVI-2) which was conducted in January 2007 and was studying the gravitropic sensing in Lens culinaris seedlings and MULTIGEN-1, performed during fall 2008, which was a "Seed-to-Seed" experiment, using Arabidopsis thaliana.

Five european EMCS experiments are planned to be flown in the next following years and are at various stages of their development:
GENARA: Gravity regulated genes in Arabidopsis thaliana
NEMATODE: Short and long-term effects of microgravity on the development of Nematodes
GRAVI-2: A follow-on of the GRAVI-1 experiment (with on-rotor fixation of the samples)
MULTIGEN-2: Gene expression regulation in Arabidopsis thaliana under microgravity
MULTIGEN-3: Observation and 3-D reconstruction of plant circumnutation

This paper will briefly introduce the EMCS facility and give an overview of the specific Experiment Unique Equipments (EUEs) hardware that has been developed, or are currently under development, for the past and future european EMCS experiments. Curent status of the Project and planned schedule will be also summarised.

EMCS, the European Modular Cultivation System, is installed in the Destiny unit of the International Space Station, ISS. In a 75 days long duration experiment called MULTIGEN-1, we recorded movements of Arabidopsis thaliana using the EMCS. One main goal in the experiment was to study possible oscillatory movements of the plant parts in weightlessness.

Plant Cultivation Chambers (PCCs) with Experimental Containers (ECs) allowed seeds to be carried into orbit, and upon experiment activation germinate when imbibed. In each PCC there were either 3 or 5 holes for the seeds to be placed in. Roots developed in a zeolite and nutrient enriched medium. White plus red light LEDs provided light in a 16:8 LD regime. The ECs were mounted on two identical rotors – four on each rotor - , allowing centrifugation in the interval 0g to 2g. Images of the plants could be provided every 5 min with the aid of cameras that viewed the plants via movable mirrors. Acceleration pulses between 0g and approximately 1g could be administered to plants, a novel feature which is possible to use in system analysis in long duration experiments.

Leaf movements of Arabidopsis thaliana (wild type) in microgravity have not been reported in the literature as far as is known to the authors. In the experiment the influence of the LD-cycle is present, but charcteristics of ultradian movements will also be presented. We have not yet analyzed all details from the about 32 000 images from the experiment. However, processing techniques for the analysis are developed and we will report on frequency and types of movements in plants in microgravity, on the EMCS rotor and under 1 g (controls).

Circumnutations in Arabidopsis have been studied on Earth and are intriguingly complicated. Under 1 g conditions the hypocotyls show a multitude of frequencies and the existence of two simultaneous oscillators generating complicated movements has been proposed. Darwin (1881) proposed an endogenous nature of the circumnutations while gravity’s possible influence has been emphasized by others researchers (review by Johnsson 1997, Mugnai et al. 2007).

We will report on the circumnutations of the plant stem as well as of side shoots in the ISS experiment. Image sequences will demonstrate circumnutations in weightlessness as well as the important rôle that rotor acceleration of about 0.8 g (g-level in the middle of the ECs) played for the amplitude and frequency of the movements. Control of the mirror position allowed us to construct 3D-images and determine rotational directions. A comparison with the short term circumnutation in Helianthus hypocotyls in Spacelab -1 will be made (Brown et al. 1990)

References: Brown, AH, Chapman, DK, Lewis RF and Vendetti AL: Circumnutations of sunflower hypocotyls in satellite orbit. Plant Physiol 94: 233 - 238. 1990.
Darwin C and Darwin F: The power of movements in plants. 1881. Reprinted Da Capo Press New York. 1966
Johnsson A.: Circumnutations: results from recent experiments on Earth and in space. - Planta 203: S 147 - S 158. 1997.
Mugnai S, Azzarello E, Masi E, Oandolfi C and Mancuso S: Nutations in plants. - In 'Rhythms in plants. Phenomenology, mechanisms, and adaptive significance' (eds. S. Manucso and S. Shabala). Pp 77 - 91. Springer-Verlag Berlin- Heidelberg. ISBN-10:3-540-68069-1. 2007.

Gravity resistance, mechanical resistance to the gravitational force, is a principal graviresponse in plants, comparable to gravitropism. Nevertheless, only limited information has been obtained for its mechanism. From ground-based experiments under centrifugal hypergravity conditions, we have hypothesized that the structural or physiological continuum of microtubule-membrane-cell wall is responsible for gravity resistance. The Resist Wall experiment aims to prove this hypothesis using microgravity conditions in space, thereby clarifying the mechanism of gravity resistance. For this purpose, we will cultivate Arabidopsis mutants defective in organization of cortical microtubules (tua6) or synthesis of membrane sterols (hmg1) as well as the wild type Columbia under microgravity and 1 g conditions on the European Modular Cultivation System (EMCS) onboard the International Space Station. These plants will be grown in the Plant Cultivation Chamber (PCC), developed for MULTIGEN-1 experiment by Dr. T.-H. Iversen, on EMCS up to reproductive stage, and their phenotypes on growth and development will be compared using video images. tua6 and hmg1 mutants are unable to form the normal cell wall and show disordered growth pattern on earth. However, it is expected that the defects of such mutants are rescued and they can grow and develop more or less normally under microgravity in space, where formation of the tough cell wall is not required. We will also analyze changes in expression of genes involved in formation of the continuum and modification of properties of related cellular components, such as the cell wall mechanical properties, under microgravity conditions, using materials fixed with RNAlater on orbit and collected to earth. The outline, scientific significance, and preliminary results of the Resist Wall experiment will be reported.

The TROPI (for tropisms) project was the first series of experiments performed on ESA's EMCS (European Modular Cultivation System) facility. The overall goal of these experiments was to gain insights into the cellular and molecular mechanisms of phototropism, the directed growth of plants in response to light, by using the model plant Arabidopsis thaliana. The first set of 8 TROPI EC's (experimental containers) with dry seeds was launched along with the EMCS facility on STS-121 in July 2006, and the second set of 16 EC's was launched on STS-115 in September 2006.

TROPI consisted of three runs, and two runs were performed in October 2006 with the third run performed in December 2006. The TROPI scientific team was at the NUSOC (Norwegian User Support and Operation Centre) in Trondheim Norway to view real time downlinks during all three runs, monitor flight operations and provide science based decisions in real time. There were a number of technical problems during all three runs. First, seed germination in space was reduced due to prolonged storage of seeds in the EC's prior to their launch.

During flight operations, incorrect commands were uploaded into the EMCS computer delaying the start of the first run. The EMCS computer also had to be rebooted several times. Due to differences between ground-based and flight model hardware, video imaging had to be reconfigured several times during the experiments. Additionally, power outages on the ISS occurred during all three runs. On the positive side, the EMCS hardware and cameras could readily be readjusted by telemetric commands, and other aspects of programming and time line also could be adjusted by telemetry. Following the TROPI experiments, the plant samples were frozen by placement into the on-orbit -80°C degree freezer (i.e., MELFI).

The frozen plant samples were returned on three space shuttle missions in 2007, and there were issues with crew procedures regarding the transfer from MELFI to the cold bags. Despite these problems, the TROPI experiment resulted in a meaningful data set in terms of video observations of tropisms and high quality RNA that can be used in gene profiling studies via microarray technology. Operational lessons learned and hardware performance issues discovered during the TROPI experiments will allow for modifications to be made by subsequent experimenters.

The GRAVI-2 experiment is planned for 2009 and will carry out in the EMCS facility. The objective of this experiment is to study the implication of amyloplasts displacement and calcium signalling in root gravisensing and, thus to understand cellular signalling mechanisms involved during the threshold acceleration. The response to gravity signal called gravitropism ensures that roots will grow down into the soil, where they take up water and nutrients, whereas shoots will grow upwards where they can photosynthesize and reproduce. In ground, the gravitropic response is obtained when a vertical seedling is re-oriented in horizontal position; its extremities (root, shoot) start bending in order to recover their normal orientation with respect to the gravity vector. Upon a gravistimulation, we observed a change in the polarity of cell perceiving gravity signal with an amyloplast sedimentation on the peripheral side. Transduction pathways are immediately activated with changes of calcium-dependant pathways. In parallel, many studies have demonstrated that cytoplasmic free Ca2+ concentration ([Ca2+] cyt) is affected by environmental stimuli. The regulation of this homeostasis involves a series of transduction events like the synthesis and activation of calcium binding and targeted proteins, like calmodulin. From results obtained in Gravi-1 experiment (D. Driss-Ecole et al., submitted), the threshold of acceleration perceived by the lentil root has been estimated to be 1.37 x 10-5 g showing that roots are strongly sensitive to gravistimulus.

In gravi-2 experiment we propose following scenario: after 21 hours of growth in microgravity environment, young seedlings will be placed on a centrifuge in order to obtain following accelerations: (1) a low acceleration, 5x10-3 g for 9 h or (2) a high acceleration 2 g for 5 min or (3) a high acceleration 2 g for a longer time (15 min). Some seedlings will be grown continuously in microgravity as control. These situations will conduct a hypothetical change in statocyte polarity and some hypothetical changes in calcium-dependant pathways. After the stimulation period, the roots will be chemically fixed to determine amyloplast distribution, free calcium distribution and calcium binding- and targeted-proteins using immunolocalisation methods. In addition to these cell analysis, we propose for understanding molecular events of gravity transduction by studying the expression of calcium-regulated genes in root cap.

The GRAVI-2 experiment hardware is accommodated in an EMCS Experiment container including two cultivation chambers, two fixative chambers and a handler unit.
We thank ESA member's team involved in the Gravi-2 development and the French Space Agency (CNES) for the financial support.

The GRAVI-1 experiment was brought on board ISS (International Space Station) by Discovery (December 2006) and carried out in January 2007 in the EMCS facility (European Modular Cultivation System).

For the first run of this experiment, lentil seedlings were hydrated and grown in microgravity for 15 h and then subjected for 13 h 40 min to centrifugal accelerations ranging from 0.29 x 10-2g to 0.99 x 10-2g. During the second run, seedlings were grown either for 30 h 30 min in microgravity (this sample was the control) or for 21 h 30 min and then subjected to centrifugal accelerations ranging from 1.2 x 10-2g to 2.0 x 10-2g for 9 h.

In both cases, root orientation and root curvature were followed by time lapse photography. Still images were downlinked in near real time to ground (N-USOC) during the experiment. The position of the root tip and the root curvature were analysed as a function of time. It has been shown that in microgravity the embryonic root curved strongly away from the cotyledons (automorphogenesis) from the 6th to the 17th h following hydration and then straightened out slowly from the 17th to the 30th h (autotropism).

Due to the autotropic straightening of roots in microgravity, their tip was oriented at an angle close to the optimal angle of curvature (120°-135°) for a period of 2 h during centrifugation. Moreover, it has been demonstrated that lentil roots grown in microgravity before stimulation were more sensitive than roots grown in 1g (Perbal et al. 2004). In these conditions, the threshold acceleration perceived by these organs was found to be comprised between 0 and 2.03 x 10-3 g and estimated punctually at 1.37 x 10-5 g by using the hyperbolic model for fitting the experimental data and by assuming that autotropism had no or little impact on the gravitropic response.

Gravisensing by statoliths should be possible at such a low level of acceleration because the actomyosin system could provide the necessary work to overcome the activation energy for gravisensing.

The "Multigen-2" has been selected by ESA as one of the European Modular Cultivation System (EMCS) experiments. In the Multigen-2 experiment Arabidopsis thaliana (Col-0) will be grown for 14 days under microgravity (static rotor) and under 1xg as the control. Samples of the seedling grown under the two gravity exposures will be analyzed using Microarray techniques post-flight on-ground to find possible differential gene expressions. The experiment will reveal genes or pathways in Arabidopsis leaves that has a function in gravitropism.

The Multigen-2 will be grown in a Cultivation Chamber (CC) on agar. The CCs will have a high relative humidity. To keep the seeds separated from conditions of high relative humidity during launch, the seeds are located on special needles in a separate chamber to prevent premature germination. The crucial part of the Multigen-2 is preservation of the seedlings at the end of the experiment run. The injection of RNALater must take place when the seedlings are still on the EMCS Rotor to preserve the samples under a controlled environment.

The Multigen-2 Science Testing Unit (STU) has been tested in the EMCS Experiment Container Development Kit (ECDK) and EMCS Experiment Reference Model (ERM) to optimization of the Multigen-2 Experiment Unique Equipment (EUE).The stowage conditions of the agar, RNALater and fixed samples have been tested. Results of these tests and description of the Multigen-2 EUE will be presented.