Gene expression analysis using microarrays has proved to be an important method in life science. The opportunity to grow higher plants on the International Space Station (ISS) opens up the possibility for gene expression profiling of plants grown in microgravity. The work presented focuses on how to meet the scientific requirements of plant growth and the sample preservation, given the technical and operational constraints associated with space research. The growth chamber (Multigen-2 Science Testing Unit) and a protocol suggested to be used in the European Modular Cultivation System (EMCS) Multigen-2 experiment on the ISS to grow and later preserve Arabidopsis seedlings, were tested on ground. The results showed that most of the plants developed normally. In order to avoid high population stress the number of seedlings per growth area should be reduced. The RNAlater preservation method to be used in the space experiment was compared with a quick freeze in Liquid Nitrogen (LN2). The RNA from samples preserved in RNAlater at room temperature for 24 h was slightly more degraded than the RNA from the LN2 preserved samples (RNA integrity number, RIN: 7.7 and 8.6, respectively). However, the RNA quality and quantity was satisfactory for microarray analysis. Of the genes analysed, 74 genes (0.28%) were significantly differentially expressed, most of them showing moderate to low regulation. Among the genes induced in the RNAlater preserved samples, three salt inducible transcription factors (ZAT10, SZF1 and SZF2) were identified, suggesting that the high salt concentration in RNAlater causes salt stress before the transcription stopped. In conclusion, the Multigen-2 preservation protocol tested here will allow for the genes regulated by microgravity in the space experiment to be revealed. The results do indicate that not all the biological processes are stopped instantly by the RNAlater. The limited diffusion indirectly caused by the microgravity may potentially result in a different degree of salt stress in the test compared to the 1 × g control during the space experiment. This has to be accounted for during the evaluation of the results. Since slightly degraded RNA was observed, further optimalisation of the preservation protocol will be performed.
Research Containing: Plant Cultivation
Reverse Genetic Approach to Exploring Genes Responsible for Cell-Wall Dynamics in Supporting Tissues of Arabidopsis thaliana under Microgravity Conditions
In 2008, the 'Cell Wall' experiment is scheduled to be launched and conducted on the International Space Station with the European Modular Cultivation System (EMCS). The main aim of this in-orbit plant science experiment is to elucidate the effect of gravitational conditions on supporting tissue formation in plants, thereby gaining new insight into the molecular mechanisms by which plants adapted to the land environment. In this first space experiment with in-orbit control experiments, we will specifically aim to elucidate the expression profiles of several candidate genes encoding proteins that are involved in the construction and restructuring of the secondary cell wall in the stem of Arabidopsis thaliana grown both in microgravity and 1G conditions. This review article deals with biological background pertinent to the 'Cell Wall' experiment, the anticipated experimental procedures to be used, together with a perspective of how this space experiment will extend our knowledge in both pure and applied life sciences.
Changes in operational procedures to improve spaceflight experiments in plant biology in the European Modular Cultivation System
The microgravity environment aboard orbiting spacecraft has provided a unique laboratory to explore topics in basic plant biology as well as applied research on the use of plants in bioregenerative life support systems. Our group has utilized the European Modular Cultivation System (EMCS) aboard the International Space Station (ISS) to study plant growth, development, tropisms, and gene expression in a series of spaceflight experiments. The most current project performed on the ISS was termed Seedling Growth-1 (SG-1) which builds on the previous TROPI (for tropisms) experiments performed in 2006 and 2010. Major technical and operational changes in SG-1 (launched in March 2013) compared to the TROPI experiments include: (1) improvements in lighting conditions within the EMCS to optimize the environment for phototropism studies, (2) the use of infrared illumination to provide high-quality images of the seedlings, (3) modifications in procedures used in flight to improve the focus and overall quality of the images, and (4) changes in the atmospheric conditions in the EMCS incubator. In SG-1, a novel red-light-based phototropism in roots and hypocotyls of seedlings that was noted in TROPI was confirmed and now can be more precisely characterized based on the improvements in procedures. The lessons learned from sequential experiments in the TROPI hardware provide insights to other researchers developing space experiments in plant biology.
The European Modular Cultivation System (EMCS) installed within the US laboratory module, Destiny, and/or the European experiment module, Columbus, onboard the International Space Station (ISS), is an ESA facility available for plant research and biological experiments. The EMCS facility uses standard experiment containers (ECs) mounted on centrifuges and provides life support such as water and gas supply systems as well as observation systems. The experiment-specific hardware such as the plant cultivation chamber, root phototropism observation chamber, and plant root gravitropism observation chamber is integrated into the EC. JAXA has five themes concerning space plant research, of which two-Cell Wall and Resist Wall-will include conducting space experiments using the EMCS facility; according to the present shuttle flight schedule, they are due to be launched in mid February 2007. The objectives of the Cell Wall / Resist Wall experiment include in-orbit growth of 10-cm-long inflorescence stems of Arabidopsis and subsequent, post-flight morphology, biological, gene expression, and cell-wall properties analyses on the ground. In this article, we describe the EMCS facility, the plant cultivation and onboard chemical fixation system. Furthermore, we also discuss the verification experiments conducted by JAXA.
The present suite of advanced space plant cultivation facilities require a significant level of resources to launch and maintain in flight. The facilities are designed to accommodate a broad size range of plant species and are, therefore, not configured to support the specific growth requirements of small plant species such as Arabidopsis thaliana at maximum efficiency with respect to mass and power. The facilities are equally not configured to support automated plant harvesting or tissue processing procedures, but rely on crew intervention and time. The recent reorganization of both spaceflight opportunities and allocation of limited in-flight resources demand that experiments be conducted with optimal efficiency. The emergence of A. thaliana as a dominant space flight model organism utilized in research on vegetative and reproductive phase biology provides strong justification for the establishment of a dedicated cultivation system for this species. This paper presents work on the design of a small plant cultivation facility directed at supporting research on the vegetative growth phase of A. thaliana . The design of the facility is based on the use of existing space flight hardware, and configured to support the fully automated germination of seed, cultivation of plants, and final termination of plant growth by chemical fixation and preservation of plant tissue.