Body of Abstract: Long-duration space missions will require plants adapted to the spaceflight environment. Limitations on growth space and down mass returned from the International Space Station (ISS) prevent many experiments from producing enough tissue for robust proteomic analyses. Transcriptional profiling has shown that plants grown during spaceflight exhibit altered molecular responses. However, changes in transcript levels do not necessarily correlate with changes in protein abundance. Instead of directly assessing protein content, Targeted Ribosome Affinity Purification (TRAP) followed by RNAseq allows for quantification of transcripts that are actively on ribosomes. These transcripts make up the translatome. The goal of the APEX-07 spaceflight experiment is to compare the transcriptome, translatome, and small RNA landscape of Arabidopsis seedlings grown on the ISS. Experimental validation of this approach was performed before the flight. Transgenic Arabidopsis expressing a FLAG epitope-tagged ribosomal protein (RPL18) were grown on PES membranes in the VEGGIE hardware for 12 days and then frozen. Parallel investigation of the transcriptome and translatome was facilitated by isolating total mRNA from frozen roots and mRNA associated with immunocaptured polysomes using a FLAG mAb. Total and polysomal mRNAs were sequenced on an Illumina NovaSeq 6000 platform and analyzed with the WySeq pipeline. Both fractions produced libraries with equivalent numbers of reads spread consistently across the lengths of all nuclear chromosomes. Of the over 25,000 loci represented in the alignment, 93% were presented in both the total and polysomal fractions. Despite these consistencies in the sequences returned from both methods, 50% of the transcripts were differentially abundant. These results show that both RNAseq and TRAP sequencing return high quality read libraries but represent unique populations of RNA. This validation shows that the transcriptomes and translatomes from APEX-07 have the power to reveal plant post transcriptional regulation in space.
Partially funded by NASA grant 80NSSC19k1481 to SEW and IP