Background: Understanding of gravity sensing and response is critical to long-term human habitation in space and can provide new advantages for terrestrial agriculture. To this end, the altered gene expression profile induced by microgravity has been repeatedly queried by microarray and RNA-seq experiments to understand gravitropism. However, the quantification of altered protein abundance in space has been minimally investigated. Results: Proteomic (iTRAQ-labelled LC-MS/MS) and transcriptomic (RNA-seq) analyses simultaneously quantified protein and transcript differential expression of three-day old, etiolated Arabidopsis thaliana seedlings grown aboard the International Space Station along with their ground control counterparts. Protein extracts were fractionated to isolate soluble and membrane proteins and analyzed to detect differentially phosphorylated peptides. In total, 968 RNAs, 107 soluble proteins, and 103 membrane proteins were identified as differentially expressed. In addition, the proteomic analyses identified 16 differential phosphorylation events. Proteomic data delivered novel insights and simultaneously provided new context to previously made observations of gene expression in microgravity. There is a sweeping shift in post-transcriptional mechanisms of gene regulation including RNA-decapping protein DCP5, the splicing factors GRP7 and GRP8, and AGO4,. These data also indicate AHA2 and FERONIA as well as CESA1 and SHOU4 as central to the cell wall adaptations seen in spaceflight. Patterns of tubulin-α 1, 3,4 and 6 phosphorylation further reveal an interaction of microtubule and redox homeostasis that mirrors osmotic response signaling elements. The absence of gravity also results in a seemingly wasteful dysregulation of plastid gene transcription.

Advances in molecular technologies have ushered in a new age of understanding across all disciplines of biology. Research in plant biology has capitalised on these novel capabilities, unlocking new understanding of how plants respond to a myriad of conditions. Plant growth in spaceflight environments represents a decades‐long endeavour, but the past 20 years have produced a wealth of data aimed toward understanding gravitational biology and informing the optimisation of plant growth for long‐duration spaceflight missions. The spaceflight environment subjects plants to low gravity and ionising radiation, which have a variety of downstream impacts on plant growth and well‐being. Omics technologies have revealed a perplexing web of stress responses to spaceflight, and optimally growing plants in these environments requires characterising the mechanistic origins of those responses. Here, we review the plant omics data gathered to date from spaceflight experiments and highlight trends and future directions as we move forward into a new age of discovery.

Plant biology experiments in microgravity face many challenges, among which are the constraints of the growth platforms available on the International Space Station (ISS). Protocols for preservation and sample return to Earth often limit efficient dissection of seedlings for downstream tissue-specific analysis. The Advanced Plant Experiment (APEx)-07 spaceflight experiment required a large quantity of dissectible, well-preserved seedlings suitable for omics analysis. During preflight tests, protocols were developed for using an agar-polyethersulfone (PES) membrane platform for seedling growth that allowed for seedling germination and growth aboard the ISS and rapid freezing to provide intact seedlings for dissection and extraction of high-quality DNA, RNA, and protein. Each component of the growth setup was carefully examined: membrane color, hydration and growth substrate, capacity for delayed germination, growth duration, harvest approach, and preservation pipelines were all individually optimized. Sterilized Arabidopsis seeds were adhered to PES membrane with guar gum. Membranes were laid onto 0.8% agar containing 0.5x Murashige and Skoog (MS) in 10 cm square Petri dishes and held at 4 °C until the experiment was actuated by placing the Petri dishes at room temperature. Seedlings were grown vertically for 12 days. PES membranes were removed from the agar, placed in the Petri dish lid, wrapped in foil, and frozen at −80 °C. Seedlings were dissected into roots and shoots and provided high-quality DNA, RNA, and protein. The system is simple, potentially adaptable for seedlings of multiple species, scalable and cost effective, and offers added versatility to existing ISS plant growth capabilities.

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