GeneTrail 3.2

Analysis of Arabidopsis thaliana exposed to different lightand gravity conditions

In order to demonstrate the ability of GeneTrail to identify potentially deregulated biological processes in plants, we here describe an analysis of a recent Arabidopsis thaliana RNA-seq data set by Herranz et al. [1]. In their study, Arabidopsis thaliana seedlings were exposed to blue light and different gravity levels in order to study the influence of both stimuli to the transcriptome. Our goal is to support their findings with enrichment analyses perfomed by GeneTrail.

Motivation

The growth of plants is affected by many environmental factors, including light, water, nutrients, and gravity. Especially for the orientation of plant roots, gravity and light are essential factors [2].

Therefore, cultivating plants in environments with lowered gravity levels, e.g., mars or a space station, might have a negative effect on plant growth. Indeed, it was shown by several groups that plants grown in lower gravity levels have a reduced ribosome biogenesis and therefore a reduced biomass production [3] [4].

In a recent study by Herranz et al. [1], Arabidopsis thaliana seedlings were grown in the International Space Station (ISS) with varying gravity levels including micro gravity, moon gravity (0.18g), mars gravity (0.36g), reduced earth gravity (0.57g), and a regular earth gravity control (1g). Additionally, seedlings were stimulated with blue light, a novel technique that tries to counteract the negative influence of a lowered gravity level on plant growth. The aim of the study is to analyze the influence of lower gravity levels on the transcriptome and on biological processes in blue light stimulated Arabidopsis thaliana seedlings.

Analysis

Preprocessing

The data set by Herranz et al. contains expression data for 17 Arabidopsis thaliana seedling samples (four micro gravity samples, three moon gravity samples, three mars gravity samples, four reduced earth gravity samples, and three control samples). The data was generated using the Illumina HiSeq2500 sequencer. The resulting paired-end reads were trimmed using Trim Galore! [5] and then aligned to an Arabidopsis thaliana STAR reference (based on the reference genome TAIR10) using STAR [6]. Afterwards, count matrices were created with RSEM [7] and normalized using DESeq2 [8]. The resulting normalized expression data set is available at the GeneLab database with accession GLDS-251.

All of these preprocessing steps were conducted by Herranz et al.

Analysis with GeneTrail

In order to identify biological processes that are affected by the change in gravity level, we performed enrichment analyses with the GeneTrail web service. To this end, we downloaded the normalized Arabidopsis thaliana data set described above from the GeneLab database. Next, we performed a GeneTrail enrichment analysis for each gravity level except for the control as follows: We uploaded the data set to the transcriptomics workflow and selected all samples of the respective gravity level as sample group. Additionally, all samples from the control gravity level were selected as reference group (for all four enrichment analyses). In order to calculate gene expression difference between the sample and the reference group, we selected the independent shrinkage t-test. As enrichment analysis method, GSEA was selected to find potentially enriched Gene Ontology (GO) and KEGG categories. The parameters used for the four enrichment analyses are listed in the tables below. Afterwards, we compared the four enrichment analysis results with the comparison functionality on the results page.

Results

The goal of our analyses is to confirm the results by Herranz et al. on ribosome biogenesis, chloroplasts and mitochondria. In this section, we discuss the different analyses and highlight supportive evidence for the findings of Herranz et al.

Ribosome biogenesis

Previous studies on Arabidopsis thaliana in low gravity levels or micro gravity levels reported a decreased ribosome biogenesis [3] [4]. In this study, the seedlings were stimulated with blue light in order to reduce this negative effect. In their analysis, Herranz et al. found several potentially enriched biological pathways related to ribosome biogenesis, which might indicate a positive influence of blue light for plant growth.

As can be seen in figure below, our GeneTrail analysis resulted in five biological processes from GO Biological Processes (GO-BP) that were consistently predicted as enriched in the different gravity levels. These processes relate to an increased ribosome biogenesis ("translation", "peptide biosynthetic process", "cellular biosynthetic process", and "biosynthetic process"), and to an increased metabolic rate ("peptide metabolic process"). These findings are supported by two consistently upregulated pathways from KEGG, which also show an increased ribosomal activity ("Ribosome") and an increased metabolic rate ("Oxidative phosphorylation"). The results for the KEGG pathways can be seen in figure below. These findings support the results from Herranz et al. and might suggest that stimulation with blue light counteracts the negative effect of low gravity levels on plant growth. A similar effect was previously achieved by stimulating Arabidopsis thaliana seedlings with red light in different gravity levels [9].

Chloroplast function

Chloroplasts are plastids in plant and algal cells that are able to conduct photosynthesis and are, hence, crucial for plant growth [10]. In previous studies of plants grown in micro gravity, the functions of chloroplasts seemed to be hampered in lower gravity levels [11] [12]. This finding was also confirmed by Herranz et al. and the results of our GeneTrail analysis, in which biological processes for chloroplast function are consistently predicted to be depleted in lower gravity levels compared to earth gravity. In total, five potentially downregulated categories related to chloroplast function were found by GeneTrail for GO Cellular Compartment (GO-CC), as shown in figure below.

Three of the five potentially downregulated categories directly relate to thylakoids, which are compartments in chloroplasts responsible for all light-stimulated reactions. The three categories are "chloroplast thylakoid", "chloroplast thylakoid membrane", and "chloroplast thylakoid lumen". Interestingly, the significance of the results consistently declines with lowering gravity level, however rises again for micro gravity.

Additionally, two categories were predicted to be downregulated in lower gravity levels. These categories are related to the structure of chloroplasts ("chloroplast envelope") and to genes in the stroma of chloroplasts ("chloroplast stroma"). These results are expected as previous studies found the shape of chloroplasts to be significantly altered under lower gravity levels and also describe a potential malfunctioning of chloroplasts in micro gravity environments [11] [12] [13].

Mitochondria

Mitochondria are compartments with a double membrane and are capable of producing ATP. However, in contrast to chloroplasts, which are also able to produce ATP, categories related to mitochondria function were predicted as enriched in lower gravity levels by Herranz et al. Indeed, this result is confirmed by the comparative enrichment analysis of GeneTrail (cf. figure below). Although not as consistent as for categories related to ribosome biogenesis, categories related to mitochondria have a decreasing significance with decreasing gravity level. An exception is the mars gravity level, for which the categories related mitochondria were lowest compared to the other gravity levels.

In summary, the enrichment results obtained from GeneTrail point towards a potential of blue light to circumvent the negative influence of lowered gravity levels on the growth of Arabidopsis thaliana seedlings. However, the interaction of gravity and light sensing on a molecular level still needs to be elucidated further in order to successfully cultivate Arabidopsis thaliana in lower gravity levels.