Characterizing Epigenetic Changes in Methylation Mutants (elp2-5 and met1-7) in Response to Spaceflight. [RNA-Seq]

Epigenetic changes in the DNA methylome are increasingly shown to play an integral role in regulating gene expression necessary for plants’ adaption to environmental stressors. Plants subjected to the novel environment of spaceflight onboard the International Space Station (ISS), show stress-related transcriptomic changes most notably associated with pathogen stress response. Here, we investigate how known terrestrial stress associated epigenetic modulations might play a role in spaceflight adaptation. To examine the role of 5mCyt in spaceflight adaptation, the APEX04-EPEX experiment conducted onboard the ISS evaluated the spaceflight altered genome wide methylation profiles of two methylation regulating gene mutants (methyltransferase 1 (met1-7) and elongator complex subunit 2 (elp2-5)) that are involved in pathogen defense response, along with a wild type Col-0 control. MethylSeq and RNAseq analyses were performed on both spaceflight grown samples and ground grown controls. In addition, the epigenetics effects that may contribute to the differential gene expression patterns observed between leaf and root tissues were also investigated in an organ-specific manner. Sterilized Arabidopsis thaliana seeds of the Columbia (Col-0), met1-7 or elp2-5 genotype were sown onto the surface of 100 mm2 square petri dishes containing a solid medium of 0.5% Phytagel™ /0.5x MS media and then wrapped in Duvetyne™ cloth. The plates were stored at 4°C and remained dormant until its installation into the Vegetable Production System (Veggie) hardware on the ISS and a comparable set installed into the Veggie hardware on the ISS Environmental Simulator (ISSES) chamber at Kennedy Space Center (KSC). These plates made up the NASA Advanced Plant Experiment 04 – Epigenetic Expression (APEX04-EPX) experiment that was launched on the SpaceX mission CRS-10. Plants in the Veggie hardware were exposed to constant light conditions of 100-135 µmoles/m2/s PAR and grown for eleven days before being harvested into Kennedy Space Center Fixation Tubes (KFTs) and fixed in RNAlater™ (Ambion, Grand Island, NY, USA). Samples from each plate were harvested into individual KFTs. The KFTs were then stowed at -80°C in the MELFI freezer aboard the ISS. The comparable ground control samples were also harvested into KFTs and stored in a standard -80°C freezer. All samples were kept frozen until delivery to the laboratory for analysis. Each plate was used as a biological replicate and a total of 4 replicates were used for the analyses. Leaf and root tissues from each sample were dissected using an Olympus microscope and materials from three individual plants per replicate were used for RNA extraction, whereas material from 10-12 plants were allocated for DNA extraction.