Arabidopsis thaliana in altered gravity: the 14-3-3κ:GFP overexpression line on the F-104 Starfighter, first flight

The increasing availability of flights on suborbital rockets creates new avenues for the study of spaceflight effects on biological systems, in particular the transitions between hypergravity and microgravity. This paper presents an initial comparison of the responses of Arabidopsis thaliana to suborbital and atmospheric parabolic flights as an important step toward characterizing these emerging suborbital platforms and their effects on biology. Transcriptomic profiling of the response of the Arabidopsis ecotype Wassilewskija (WS) to the aggregate spaceflight experiences in the Blue Origin New Shepard and Virgin Galactic SpaceShipTwo rockets revealed that the transcriptomic load induced by flight differed greatly between the two flights, yet was biologically related to traditional parabolic flight responses. The sku5 skewing mutant and 14-3-3κ:GFP regulatory protein overexpression lines each showed altered intra-platform responses compared to WS in the Blue Origin and parabolic flights, respectively. An additional parabolic flight using the F-104 Starfighter showed that the response of 14-3-3κ:GFP to flight was modulated in a similar manner to the WS line. Despite the differing genotypes, experimental workflows, flight profiles and platforms, alteration of gene expression remodeling central metabolic processes was commonly observed as a response to the flights. The processes included carbon and nitrogen metabolism, branched-chain amino acid degradation, and hypoxic responses. The timing and directionality of differentially-expressed genes involved in the conserved pathways differed among the platforms. The data presented herein highlight the potential for various suborbital platforms to contribute insights into biological responses to spaceflight, and further suggest that in-flight fixation during suborbital experiments will provide insights into responses to each phase of flight. 10-day-old seedlings overexpressing 14-3-3κ:GFP, from a WS background, were transferred from 0.5x MS media plates to three Kennedy Space Center fixation tubes (KFTs), which were sealed. KFTs were actuated in-flight, exposing the seedlings to RNAlater, following the transition out of microgravity and about 1.5 min after the return to level flight. A ground control KFT was also actuated during the flight. Seedlings and RNAlater were transferred from KFTs to 50 mL Falcon tubes. Roots from fixed seedlings were dissected from shoots. Whole roots were separated into three biological replicates for each condition. Total RNA was extracted from roots comprising each replicate and submitted for microarray analysis. The Affymetrix GeneChip Arabidopsis Gene (AraGene) 1.0 ST array was used to measure gene expression levels. The transcriptomic data from the flight-actuated samples were then compared to those of the ground controls in order to identify differentially-expressed genes between the ground control and the two flight conditions.