RNA-seq analyses of Arabidopsis thaliana seedlings after exposure to blue-light phototropic stimuli in microgravity.

PREMISE: Plants synthesize information from multiple environmental stimuli when determining their direction of growth. Gravity, being ubiquitous on Earth, plays a major role in determining the direction of growth and overall architecture of the plant. Here, we utilized the microgravity environment on board the International Space Station (ISS) to identify genes involved influencing growth and development of phototropically stimulated seedlings of Arabidopsis thaliana. METHODS: Seedlings were grown on the ISS, and RNA was extracted from 7 samples (pools of 10-15 plants) grown in microgravity (µg) or Earth gravity conditions (1-g). Transcriptomic analyses via RNA sequencing (RNA-seq) of differential gene expression was performed using the HISAT2-Stringtie-DESeq2 RNASeq pipeline. Differentially expressed genes were further characterized by using Pathway Analysis and enrichment for Gene Ontology classifications. RESULTS: For 296 genes that were found significantly differentially expressed between plants in microgravity compared to 1-g controls, Pathway Analysis identified eight molecular pathways that were significantly affected by reduced gravity conditions. Specifically, light-associated pathways (e.g., photosynthesis-antenna proteins, photosynthesis, porphyrin, and chlorophyll metabolism) were significantly downregulated in microgravity. CONCLUSIONS: Gene expression in A. thaliana seedlings grown in microgravity was significantly altered compared to that of the 1-g control. Understanding how plants grow in conditions of microgravity not only aids in our understanding of how plants grow and respond to the environment but will also help to efficiently grow plants during long-range space missions.

Spaceflight-related suboptimal conditions can accentuate the altered gravity response of Drosophila transcriptome.

Genome-wide transcriptional profiling shows that reducing gravity levels during Drosophila metamorphosis in the International Space Station (ISS) causes important alterations in gene expression: a large set of differentially expressed genes (DEGs) are observed compared to 1g controls. However, the preparation procedures for spaceflight and the nonideal environmental conditions on board the ISS subject the organisms to additional environmental stresses that demonstrably affect gene expression. Simulated microgravity experiments performed on the ground, under ideal conditions for the flies, using the random position machine (RPM), show much more subtle effects on gene expression. However, when the ground experiments are repeated under conditions designed to reproduce the additional environmental stresses imposed by spaceflight procedures, 79% of the DEGs detected in the ISS are reproduced by the RPM experiment. Gene ontology analysis of them shows they are genes that affect respiratory activity, developmental processes and stress-related changes. Here, we analyse the effects of microgravity on gene expression in relation to the environmental stresses imposed by spaceflight. Analysis using 'gene expression dynamics inspector' (GEDI) self-organizing maps reveals a subtle response of the transcriptome to microgravity. Remarkably, hypergravity simulation induces similar response of the transcriptome, but in the opposite direction, i.e. the genes promoted under microgravity are usually suppressed under hypergravity. These results suggest that the transcriptome is finely tuned to normal gravity and that microgravity, together with environmental constraints associated with space experiments, can have profound effects on gene expression.

Identification and localization of a nucleolin homologue in onion nucleoli.

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

Immunolocalization of DNA at nucleolar structural components in onion cells.

Intranucleolar DNA, including ribosomal DNA (rDNA), was localized in situ in proliferating onion cells under the electron microscope using an anti-DNA monoclonal antibody and a postembedding indirect immunogold procedure. In the interphase nucleolus of this species, characterized by a very high amount of rRNA genes, we found DNA concentrated mostly in fibrillar centres (FCs) and in the region of the dense fibrillar component (DFC) immediately surrounding them. Clusters of gold particles were frequently seen covering both of these structural components of the nucleolus at the same time. Moreover, the same technique, applied to transcriptionally arrested quiescent onion cells, showed the nucleolar DFC devoid of DNA. Also, in mitotic cells at telophase, the prenucleolar material, which has the same morphological and cytochemical features as the DFC, does not contain DNA. These data suggest the existence of at least two subcomponents of the DFC in the onion cell nucleolus, one associated with pre-rRNA synthesis, and the other, with further processing of transcripts, already released from the rDNA template. We conclude that the first subcomponent forms part of the "transition between FC and DFC", which is the in situ structural counterpart of pre-rRNA synthesis. This transition is morphologicaly sizeable in onion cells, because of their high number of rRNA genes and the large size of the DFC mass; however, it would be largely detectable in situ in other cell systems, where the whole DFC comprises only a thin layer and the amount of rDNA is considerably reduced.