Isabel Matía

Plant cell proliferation and growth are altered by microgravity conditions in spaceflight.

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period.

Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

The "Root" experiment of the "Cervantes" Spanish Soyuz Mission: cell proliferation and nucleolar activity alterations in Arabidopsis roots germinated in real or simulated microgravity

In an experiment conducted in the “Cervantes” Spanish Soyuz Mission, a 10-day flight to the International Space Station, Arabidopsis seeds were germinated, seedlings grew for 4 days at 22ºC, and they were fixed in flight with paraformaldehyde. A ground 1 g control experiment was replicated, and an additional experiment in simulated microgravity, using a Random Positioning Machine, was performed in the same conditions. Structural, morphometric and immunocytochemical data were compared. Glutaraldehyde-fixed seedlings similarly grown in the Space Shuttle (STS-84 Mission) were also used for ultrastructural and morphometric studies. Seedlings grown for 4 days in real or simulated weightlessness showed a longer size than the ground 1 g control. Root meristematic cells showed an enhanced proliferating rate, but ribosome biogenesis was reduced, as inferred from the nucleolar size and from the levels of the nucleolar protein nucleolin. This could be the consequence of the acceleration of the cell cycle, with shortening of its phases. Weightlessness induces stress in the plant, influencing cellular processes decisive for development and morphogenesis. This stress may endanger the plant and would require the action of compensating specialized defence mechanisms.

Functional alterations of root meristematic cells of Arabidopsis thaliana induced by a simulated microgravity environment

Environmental gravity modulates plant growth and development, and these processes are influenced by the balance between cell proliferation and differentiation in meristems. Meristematic cells are characterized by the coordination between cell proliferation and cell growth, that is, by the accurate regulation of cell cycle progression and the optimal production of biomass for the viability of daughter cells after division. Thus, cell growth is correlated with the rate of ribosome biogenesis and protein synthesis. We investigated the effects of simulated microgravity on cellular functions of the root meristem in a sequential study. Seedlings were grown in a clinostat, a device producing simulated microgravity, for periods between 3 and 10days. In a complementary study, seedlings were grown in a Random Positioning Machine (RPM) and sampled sequentially after similar periods of growth. Under these conditions, the cell proliferation rate and the regulation of cell cycle progression showed significant alterations, accompanied by a reduction of cell growth. However, the overall size of the root meristem did not change. Analysis of cell cycle phases by flow cytometry showed changes in their proportion and duration, and the expression of the cyclin B1 gene, a marker of entry in mitosis, was decreased, indicating altered cell cycle regulation. With respect to cell growth, the rate of ribosome biogenesis was reduced under simulated microgravity, as shown by morphological and morphometric nucleolar changes and variations in the levels of the nucleolar protein nucleolin. Furthermore, in a nucleolin mutant characterized by disorganized nucleolar structure, the microgravity treatment intensified disorganization. These results show that, regardless of the simulated microgravity device used, a great disruption of meristematic competence was the first response to the environmental alteration detected at early developmental stages. However, longer periods of exposure to simulated microgravity do not produce an intensification of the cellular damages or a detectable developmental alteration in seedlings analyzed at further stages of their growth. This suggests that the secondary response to the gravity alteration is a process of adaptation, whose mechanism is still unknown, which eventually results in viable adult plants.