Cell cycle acceleration and changes in essential nuclear functions induced by simulated microgravity in a synchronized Arabidopsis cell culture

Abstract

Zero gravity is an environmental challenge unknown to organisms throughout evolution on Earth. Nevertheless, plants are sensitive to altered gravity, as exemplified by changes in meristematic cell proliferation and growth. We found that synchronized Arabidopsis-cultured cells exposed to simulated microgravity showed a shortened cell cycle, caused by a shorter G2/M phase and a slightly longer G1 phase. The analysis of selected marker genes and proteins by quantitative polymerase chain reaction and flow cytometry in synchronic G1 and G2 subpopulations indicated changes in gene expression of core cell cycle regulators and chromatin-modifying factors, confirming that microgravity induced misregulation of G2/M and G1/S checkpoints and chromatin remodelling. Changes in chromatin-based regulation included higher DNA methylation and lower histone acetylation, increased chromatin condensation, and overall depletion of nuclear transcription. Estimation of ribosome biogenesis rate using nucleolar parameters and selected nucleolar genes and proteins indicated reduced nucleolar activity under simulated microgravity, especially at G2/M. These results expand our knowledge of how meristematic cells are affected by real and simulated microgravity. Counteracting this cellular stress is necessary for plant culture in space exploration.

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

“…In contrast with the downregulation of phototropism and related biosynthetic and metabolic pathways, we found two very clearly upregulated functions in microgravity‐grown seedlings when compared to 1‐ g spaceflight samples: ribosome biosynthesis and oxidative phosphorylation (Appendices S16 and S17). In both true microgravity (Matía et al., ) and different simulated microgravity facilities using both seedlings and cell cultures (Manzano et al., ; Kamal et al., ), ribosome biogenesis was reduced. Our recent spaceflight results showed that red light can compensate for this effect (Valbuena et al., ), particularly by increasing cell growth (measured by means of ribosome biosynthesis in the nucleolus) that was depleted without light stimulation.…”

“…In terms of cell growth and cell proliferation, it has been shown that when seedlings are grown in darkness, there is a lack of balance between these key plant development functions in microgravity (Matía et al., ). Further evidence for this observation was provided by analyzing a dark‐grown, synchronized cell culture grown in simulated microgravity (Kamal et al., ). Recent spaceflight results show that red light can compensate for this effect (Valbuena et al., ), particularly increasing cell growth (measured by means of ribosome biosynthesis in the nucleolus) that was depleted without light stimulation.…”

Differential transcriptional profile through cell cycle progression in Arabidopsis cultures under simulated microgravity

“…These reference times have been confirmed by flow cytometry in a parallel experiment with aliquots of the same cell culture system and microgravity simulator conditions as described here [28]. Cells were recovered from the agorose and frozen until RNA extraction.…”

“…Since we had previously established that there was a quick entry into M phase under simulated microgravity conditions [28], agarose-embedded cultures (synchronous and asynchronous subpopulations) were exposed to simulated microgravity conditions for different experimental durations accordingly. To obtain the maximum enrichment in each cell cycle subpopulation (higher than 2/3 of the cell population), the exposure time was 7h RPM / 10h 1g control hours for the G2/M synchronized samples, 14h RPM / 16h 1g control for the G1 synchronized cells and 14h RPM / 14h 1g control for the asynchronous reference cells.…”

“…The simulated microgravity methodology has already been validated in this cell culture system [26,27], and an effect on cell cycle regulation was observed in non-synchronous populations [25]. To reveal the transcriptional mechanisms of the plant response to the simulated microgravity environment, we performed a microarraybased transcriptomic approach on immobilized and synchronized cell cultures (enriched in G2/M and G1 cells as described in [28]) grown under simulated microgravity conditions produced in a Random Positioning Machine (RPM). This allowed us to confirm and extend previously described alterations in cell cycle and to provide new insights into the stress pathways involved in the response to altered gravity conditions.…”