Spaceflight induces novel regulatory responses in Arabidopsis seedling as revealed by combined proteomic and transcriptomic analyses

Abstract

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.

Conclusions: The datasets gathered from Arabidopsis seedlings exposed to microgravity revealed marked impacts on post-transcriptional regulation, cell wall synthesis, redox/microtubule dynamics, and plastid gene transcription. The impact of post-transcriptional regulatory alterations represents an unstudied element of the plant microgravity response with the potential to significantly impact plant growth efficiency and beyond. What's more, addressing the effects of microgravity on AHA2, CESA1, and alpha tubulins has the potential to enhance cytoskeletal organization and cell wall composition, thereby enhancing biomass production and growth in microgravity. Finally, understanding and manipulating the dysregulation of plastid gene transcription has further potential to address the goal of enhancing plant growth in the stressful conditions of microgravity.

Keywords: Arabidopsis; Gravitropism; Gravity; Phosphorylation; Plastid; Post-transcriptional gene regulation; Proteomics; Spaceflight; Transcriptomics.

Fig. 1

Overview of transcripts (top) and proteins (bottom) differentially expressed during spaceflight. RNA sequencing and protein mass spectrometry was performed on space-flown Arabidopsis seedlings and compared to ground controls. The number adjacent to each sector label indicates the count of genes/proteins that occupy the slice. Subcellular localizations are shown in the outermost circles. SUBA4 [13] (V.4.6.0, accessed June 2017) was used to identify the subcellular localizations. Gene/protein accessions and expression values are provided in Supplemental File 1

Fig. 2

Heat map of a gene ontology enrichment analysis of genes/proteins differentially upregulated in space (left) or upregulated on Earth (right). The central callouts list the differentially expressed genes/proteins within the selected GO term. Enrichment was performed for GO terms for biological processes using Orange (V.3.7.0, GO terms were accessed July 2017). Only transcripts with a log-fold change ±1 (FDR ≤ 0.05) or proteins with a log-fold change ±0.2 (P ≤ 0.05) were included in the analysis. The upregulated and downregulated genes were run separately, and the resultant GO enrichments were thinned to include GO terms with a p-value ≤0.05 using the Bonferroni correction for multiple comparisons. Numbers next to the terms represent the number of genes or proteins that appeared in that term, followed by the total number genes or proteins associated with that term. Supporting data can be found in Supplemental File 2

Fig. 3

Genes/proteins differentially expressed in both transcript and protein datasets. Of the 17 genes, 12 have coordinated expression patterns (i.e. increased or decreased in both the transcript and protein data), while 5 genes exhibit opposing expression patterns between datasets. X-axis = transcript expression, Y-axis = protein expression. Boxes indicated gene/protein name of the associated point. Some of the most extreme expression patterns highlight TONOPLAST INTRINSIC PROTEIN 3–2 (AT1G17810), a cofactor-dependent phosphoglycerate mutase (dPGM, AT5G04120), and a DNA-polymerase epsilon catalytic subunit A (AT1G19530) within the datasets

Fig. 4

Summary of the results and insight provided from the transcript (RNA), protein, and post-translational modification (PTM) datasets. Transcript expression profiles were characterized by RNA-seq and protein expression profiles were characterized and quantified by iTRAQ LC-MS/MS. Expression and fragmentation data characterized the regulatory dynamics induced by adaptation to the spaceflight environment in Arabidopsis thaliana. Gene Ontology analysis identified PTR, PTM, and Degradation candidate genes. DEGs = differentially expressed genes, DAPs = differentially abundant proteins, PTR = post-transcriptional regulation