With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility, and reusability of pipeline data; to provide a template for data processing of future spaceflight-relevant datasets; and to encourage cross-analysis of data from other databases with the data available in GeneLab.

Introduction: Traveling to nearby extraterrestrial objects having a reduced gravity level (partial gravity) compared to Earth’s gravity is becoming a realistic objective for space agencies. The use of plants as part of life support systems will require a better understanding of the interactions among plant growth responses including tropisms, under partial gravity conditions. Materials and Methods: Here, we present results from our latest space experiments on the ISS, in which seeds of Arabidopsis thaliana were germinated, and seedlings grew for six days under different gravity levels, namely micro-g, several intermediate partial-g levels, and 1g, and were subjected to irradiation with blue light for the last 48 h. RNA was extracted from 20 samples for subsequent RNAseq analysis. Transcriptomic analysis was performed using the HISAT2-Stringtie-DESeq pipeline. Differentially expressed genes were further characterized for global responses using the GEDI tool, gene networks and for Gene Ontology (GO) enrichment. Results: Differential gene expression analysis revealed only one differentially expressed gene (AT4G21560, VPS28-1 a vacuolar protein) across all gravity conditions using FDR correction (q < 0.05). However, the same 14 genes appeared differentially expressed when comparing either micro-g, low-g level (< 0.1g) or the Moon g-level with 1g control conditions. Apart from these 14-shared genes, the number of differentially expressed genes was similar in microgravity and the Moon g-level and increased in the intermediate g-level (< 0.1g), but it was then progressively reduced as the difference with the Earth gravity became smaller. The GO groups were differentially affected at each g-level: light and photosynthesis GO under microgravity, genes belonged to general stress, chemical and hormone responses under low-g, and a response related to cell wall and membrane structure and function under the Moon g-level. Discussion: Transcriptional analyses of plants under blue light stimulation suggests that root blue-light phototropism may be enough to reduce the gravitational stress response caused by the lack of gravitropism in microgravity. Competition among tropisms induces an intense perturbation at the micro-g level, which shows an extensive stress response that is progressively attenuated. Our results show a major effect on cell wall/membrane remodeling (detected at the interval from the Moon to Mars gravity), which can be potentially related to graviresistance mechanisms.

The response of plants to the spaceflight environment and microgravity is still not well understood, although research has increased in this area. Even less is known about plants’ response to partial or reduced gravity levels. In the absence of the directional cues provided by the gravity vector, the plant is especially perceptive to other cues such as light. Here, we investigate the response of Arabidopsis thaliana 6-day-old seedlings to microgravity and the Mars partial gravity level during spaceflight, as well as the effects of red-light photostimulation by determining meristematic cell growth and proliferation. These experiments involve microscopic techniques together with transcriptomic studies. We demonstrate that microgravity and partial gravity trigger differential responses. The microgravity environment activates hormonal routes responsible for proliferation/growth and upregulates plastid/mitochondrial-encoded transcripts, even in the dark. In contrast, the Mars gravity level inhibits these routes and activates responses to stress factors to restore cell growth parameters only when red photostimulation is provided. This response is accompanied by upregulation of numerous transcription factors such as the environmental acclimation-related WRKY-domain family. In the long term, these discoveries can be applied in the design of bioregenerative life support systems and space farming.

Clinorotation was the first method designed to simulate microgravity on ground and it remains the most common and accessible simulation procedure. However, different experimental settings, namely angular velocity, sample orientation, and distance to the rotation center produce different responses in seedlings. Here, we compare A. thaliana root responses to the two most commonly used velocities, as examples of slow and fast clinorotation, and to vertical and horizontal clinorotation. We investigate their impact on the three stages of gravitropism: statolith sedimentation, asymmetrical auxin distribution, and differential elongation. We also investigate the statocyte ultrastructure by electron microscopy. Horizontal slow clinorotation induces changes in the statocyte ultrastructure related to a stress response and internalization of the PIN-FORMED 2 (PIN2) auxin transporter in the lower endodermis, probably due to enhanced mechano-stimulation. Additionally, fast clinorotation, as predicted, is only suitable within a very limited radius from the clinorotation center and triggers directional root growth according to the direction of the centrifugal force. Our study provides a full morphological picture of the stages of graviresponse in the root tip, and it is a valuable contribution to the field of microgravity simulation by clarifying the limitations of 2D-clinostats and proposing a proper use.

Understanding plant adaptive responses to the space environment is a requisite for enabling space farming. Spaceflight produces deleterious effects on plant cells, particularly affecting ribosome biogenesis, a complex stress-sensitive process coordinated with cell division and differentiation, known to be activated by red light. Here, in a series of ground studies, we have used mutants from the two Arabidopsis nucleolin genes (NUC1 and NUC2, nucleolar regulators of ribosome biogenesis) to better understand their role in adaptive response mechanisms to stress on Earth. Thus, we show that nucleolin stress-related gene NUC2 can compensate for the environmental stress provided by darkness in nuc1 plants, whereas nuc2 plants are not able to provide a complete response to red light. These ground control findings, as part of the ESA/NASA Seedling Growth spaceflight experiments, will determine the basis for the identification of genetic backgrounds enabling an adaptive advantage for plants in future space experiments.