Spaceflight-induced alternative splicing during seedling development in Arabidopsis thaliana

Abstract

Plants grown in spaceflight experience novel environmental signals, including those associated with microgravity and ionizing radiation. Spaceflight triggers a response involving transcriptional re-programming and altered cell morphology, though many aspects of this response remain uncharacterized. We analyzed the spaceflight-induced transcriptome with a focus on genes that undergo alternative splicing to examine differential splicing associated with spaceflight-an unstudied characteristic of the molecular response to spaceflight exposure. RNA sequence data obtained during the APEX03 spaceflight experiment that was collected from two Arabidopsis thaliana ecotypes at two seedling stages grown onboard the International Space Station, or as ground controls at Kennedy Space Center, were re-examined to detect alternative splicing differences induced by spaceflight. Presence/absence variation analysis was used to identify putative expression-level differences in alternatively spliced isoforms between spaceflight and ground controls and was followed by analysis of significant differential alternative splicing. This study provides the first evidence of a role for alternative splicing in the molecular processes of physiological adaptation to the spaceflight environment.

Fig. 1

Detection of spaceflight-induced alternatively spliced transcript isoforms. a Bioinformatics pipeline utilized to identify all transcript isoforms and significantly differentially spliced genes present in the original RNA sequencing dataset. Steps inside the dashed outline were performed on each individual replicate. See Methods for more detail on gtf merging. b Comparison of alternatively spliced isoforms present or absent in spaceflight in both ecotypes and developmental ages tested. c Count of differentially alternatively spliced genes and the associated count of transcript isoforms observed in each condition. d Comparison of the differentially alternatively spliced genes observed in the Columbia-0 (Col-0) or the Wassilewskija (WS) ecotype background. Differentially alternatively spliced genes observed at both 4- and 8-day time points in either ecotype background were pooled for this analysis. (C Columbia-0, W Wassilewskija, 4 4 days of age, 8 8 days of age. C4 Columbia-0 4 days of age, W8 Wassilewskija 8 days of age)

Fig. 2

Visualization of switch in isoform ratio between spaceflight and ground controls for gene AT2G33770. a Screenshot from Integrative Genomics Viewer displaying sequencing coverage spanning the AT2G33770 gene model in spaceflight and ground control samples from either the 4-day Columbia-0 (Col-0) or 8-day Wassilewskija (WS) backgrounds. Asterisks indicate exons within the gene model solely present in transcript isoform TCONS_0029096. Arrows indicate lack of sequencing coverage in spaceflight samples corresponding to the unique exons in TCONS_0029096. Small numbers in the upper left hand corner of each panel denote the range of the y axis scale for each condition. b Isoform ratios in spaceflight and ground controls for transcript isoforms TCONS_0029094, TCONS_0029095, and TCONS_0029096 associated with gene AT2G33770 in Col-0 4-day and WS 8-day backgrounds. Isoform ratio was calculated by FPKM (fragments per kilobase of transcript per million mapped reads)