Instructor, University of Wisconsin, Madison WI
In order for humans to become a multi-planetary species there are many technological and biological hurdles that must be overcome. The closest two potential sites for extraterrestrial colonization (apart from low earth orbit) are the moon and mars. Both of these potential first steps in the colonization of our solar system have enough mass to exhibit approximately half the gravitational force of earth. Thus, if human kind is to create sustainable extra-terrestrial communities, then our species will have to select traits in our companion organism that allow them to adapt to these novel alterations in the gravitational field. Here we describe the transcriptomic response of four different natural varieties of the eukaryote Arabidopsis thaliana (Col-0, Ler-0, Cvi-0, WS-2) to micro-gravity as experienced during space flight. Sequencing of the mRNA extracted from these plants after 8 days in orbit has provided a unique insight into how plant transcriptomes respond to the stresses of space flight and micro-gravity.
Here we present the RNA-seq data from 4 natural Arabidopsis accessions grown on Earth or in low Earth Orbit upon the International Space Station. This essentially allows 2 types of experiments to be conducted. In the first one we can compare how each different variety responds to the stresses of spaceflight. Alternatively these data can be used to examine how natural genomic diversity affects gene expression by simply comparing the ground control samples or the space-flown samples against one another.
Seeds were grown on 60mm circular Petri dishes containing 1% (w/v) Phytagel, 0.3% (w/v) Sucrose and ½ strength Epstein salts and then treated with 100 nmol m-2 white light for 12 hours. The samples were then placed in PDFUs (Petri Dish Fixation Units) inside a BRIC (Biological Research In Canister). They were then sent into orbit on the SpaceX Falcon 9 rocket which released a Dragon capsule that delivered samples to the International Space Station. The seedlings were then grown for 8 days in darkness at 22˚C before being submerged in Ambion RNAlater and frozen at -80oC. The samples were then returned to the Kennedy Space Center where they were removed from the BRIC and then returned to the UW Madison on dry ice. 4 biological repeats were harvested for two of the ecotypes (Col-0 and Ws-2) and 3 for the other 2 *Ler-0 and Cvi-0). Samples were pulverized using an automated sample grinder and RNA was extracted using RNeasy Plant Mini Kit (Qiagen) and DNA was removed using the Ambion TURBO DNase kit. Total RNA underwent rRNA reduction using the Illumina Ribo-Zero plant leaf kit and the library was created using the Illumina TruSeq RNA sample preparation kit. One 100 bp, paired-end, unstranded, 2x100bp read using version 3 SBS chemistry, Illumina Next Generation sequencing was performed using a HiSeq2000 (Illumina) by the University of Wisconsin-Madison Sequencing Facility. The HiSeq2000 sequencing instrument used CASAVA 1.8.2 as the base caller software. Two technical repeats for each sample were performed for each sequencing run. A parallel set of plants were grown under identical conditions in the International Space Station Environmental Simulator at Kennedy Space Center. These were harvested, processed and sequenced identically to the flight samples and constituted the ground controls for the experiment.
Many previous space flight experiments have been conducted to investigate plant responses to micro-gravity and the environmental factors associated with growth in space. These experiments have been conducted in many different species and ecotypes. These data aim to explore how insightful comparisons of these different experiments can be by investigating the conserved and unique patterns between ecotypes. A set of core spaceflight response genes was identified and their biological function predicted using the DAVID functional annotation database and QTL-netminer literature and interactome mining tools. These predictions were then investigated by performing qRT-PCR on RNA samples from both the spaceflight experiment and ground based hypoxia and/or hydrogen peroxide response experiments.
UW-Madison Botany department currently has an experimental new teaching system based in the Co-laboratory. This space is designed to promote inter-disciplinary collaboration between botanists, geneticists, statisticians, engineers, computer programmers and rocket Scientists. Students learn new skills by performing experiments designed to test predictions generated using the NASA GeneLab natural variation experiment. The students are introduced to NGS technologies by research scientists and are then encouraged to explain it other students from different departments. Students that specialize in computer science are encouraged to explore the RNAseq data using the DNA subway. Students that are interested in molecular biology create mutants and test the bioinformatic predictions. Engineering students then design, build, test and refine new methods to investigate plant responses to environmental stress that resemble those experienced during spaceflight. Graduate students can be provided with the raw sequencing data in the iPlant Discovery Environment for more complex analysis.
The weGAS and iGAS protocols will be provided after publication