Meristematic cell proliferation and ribosome biogenesis are decoupled in diamagnetically levitated Arabidopsis seedlings

Abstract

Background: Cell growth and cell proliferation are intimately linked in the presence of Earth's gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter.

Results: We studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells.

Conclusions: In our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.

Figure 1

Experimental set up and seedling growth. A) Three samples were exposed simultaneously to the magnetic field, in three different positions within the magnet bore: 0 g*, 1 g* and 2 g* tubes. An additional sample was maintained under the same conditions, except for the magnetic field, outside the magnet (1 g control). B) Effective gravity acting on water within the 0 g* tube. The colour scale indicates the magnitude of the effective gravity as percent of the ground gravity g. Arrows show the magnitude and direction of the effective gravity vector. C) Image taken of the sample tube located at the 0 g* position, after the 4-day experiment. After growing in darkness, seedlings show a clear orientation according to the gravity vector.

Figure 2

The root columella cells observed by phase-contrast microscopy. (Left-hand image) Samples were fixed, embedded in resin and 2 μm semithin sections were obtained from them and observed under phase-contrast light microscopy. Those samples grown under magnetic levitation (0 g* tube) for 4 days show the same distribution of statoliths (indicated by arrows) as in control 1 g samples (right-hand image) grown outside the magnet. The statoliths collect near the basal membrane of the columella cells, under the force of gravity; the arrow between the images indicates the direction of gravity.

Figure 3

Morphometric parameters of seedlings. Diagram of seedling and root length in the two-day (A) and four-day long (B) experiments. Statistically significant differences in length (p < 0.05), compared to the control, are indicated with a ‘§’ symbol when compared with the 1 g external control, and with ‘#’ when compared with the 1 g* control. The most pronounced differences were found in the 0 g* sample, which shows significant differences with respect to both external and internal controls in the two experiments. Seedlings in the internal and external controls (1 g and 1 g*) show significant differences after 4 days’ growth, but not after 2 days. After 4 days, all samples grown within the magnet (0 g*, 1 g* and 2 g*) are significantly different from the external control (1 g).

Figure 4

Rate of local cell production (Number of cells/mm in each cell row of the root meristem). A) Two-day experiment. B) Four-day experiment. Statistically significant differences in cell production (p < 0.05), compared to the control, are indicated with a ‘§’ symbol when compared with the 1 g external control, and with ‘#’ when compared with the 1 g* control. Seedlings grown in the 0 g* tube show consistently higher cell proliferation rates compared with controls.

Figure 5

Average size of the nucleolus (cross-sectional area in μm²) and percentage of granular component (GC). A) Two-day experiment. B) Four-day experiment. Statistically significant differences in nucleolar size (p < 0.05), compared to the control, are indicated with a ‘§’ symbol when compared with the 1 g external control, and with ‘#’ when compared with the 1 g* control. Seedlings grown in the 0 g* tube show consistently a smaller nucleolus size. C) Representative electron microscope images of nucleoli from the 4-day-long experiment. Even though the nucleolus is clearly smaller in samples from the 0 g* tube, compared to the external control, the proportion and distribution of the different ultrastructural components of the nucleolus is similar in both conditions. GC: granular component. DFC: dense fibrillar component. Arrows indicate fibrillar centres.

Figure 6

Immuno-gold electron microscopical detection of the nucleolar protein nucleolin. A) Portions of nucleoli from root meristematic cells, labelled with anti-nucleolin antibody and visualised with colloidal gold particles, are shown for the 2-day experiment: (left) 0 g* samples, (right) external 1 g control samples. Nucleolin is localised in the dense fibrillar component (DFC), surrounding fibrillar centres (arrows) in both cases. The 0 g* sample shows a lower density of gold particles, indicating a lower nucleolus activity. B and C) Density of immunogold particles measured after two- (B) and four-day long (C) experiments. Statistically significant differences in density (p < 0.05), compared to the control, are indicated with a ‘§’ symbol when compared with the 1 g external control, and with ‘#’ when compared with the 1 g* control.

Figure 7

Cyclin B1 expression in root tips revealed by GUS staining. The use of the reporter gene line CYCB1:GUS allowed the microscopical visualization of the expression of the cyclin B1 gene. Whole mount preparations of roots were stained and observed by optical microscopy. A) 2 days’ and B) 4 days’ growth; from left to right: 1 g external control, 0 g*, 1 g* and 2 g*. C) and D) Quantitative study of the expression of the cyclin B1 gene by measuring the integrated optical density (I.O.D.) in the two-day (C) and in the four-day (D) experiments. Statistically significant differences in IOD (p < 0.05), compared to the control, are indicated with a ‘§’ symbol when compared with the 1 g external control, and with ‘#’ when compared with the 1 g* control. GUS staining show an overall decrease in the expression of this gene (which is usually considered to be a marker of cell proliferation)in the magnet. This is especially clear in samples from the simulated microgravity position (0 g*).

Figure 8

Auxin distribution in root tips revealed by GUS staining. The use of the reporter gene line DR5:GUS allowed the microscopical visualization of the auxin distribution. Whole mount preparation of roots were stained and observed by optical microscopy. A) Optical microscopy images of DR5-GUS-stained root meristems from seedlings grown for 4 days in the magnet; the staining shows the distribution of auxin in the root tip. From top-left to bottom right: samples from the 1 g external control, 0 g*, 1 g* and 2 g* positions in the magnet. B) Quantitative study of the GUS staining by measuring the integrated optical density (I.O.D.) in the four-day experiment. Statistically significant differences in IOD (p < 0.05), compared to the control, are indicated with a ‘§’ symbol when compared with the 1 g external control, and with ‘#’ when compared with the 1 g* control.