Mechanical stress in Arabidopsis leaves orients microtubules in a 'continuous' supracellular pattern

Abstract

Background: Cortical microtubules form a dynamic network and continuously undergo shrinking (catastrophe), pausing and rebuilding (rescue). The advantage of such a dynamic system is that it may mediate appropriate responses in a short time span. Microtubules are known to play a pivotal role in determining the orientation of the cellulose microfibril deposition in the plant cell wall. The latter is a solid exoskeleton surrounding the protoplast. It forms the physical framework that interconnects most cells and has to bear the tensile stresses within the tissue. Here we describe the effect of externally applied pressure on microtubule organization in growing Arabidopsis leaves.

Results: Confocal microscopy examination of transgenic plants bearing GFP-tagged TUA6 proteins led to the observation that application of an additional mechanical pressure on growing Arabidopsis leaves triggers an excessive bundling of microtubules within the individual cell. Besides, the microtubules seem to align in neighboring cells, creating a 'continuous' supracellular pattern. This effect occurs within 3 hours after applied external force and is age-dependent, whereby only cells of leaves up to 19 days after sowing (DAS) are susceptible to the applied pressure.

Conclusions: Upon externally applied pressure on developing Arabidopsis leaves, microtubules bundle and rearrange to form seemingly continuous supracellular patterns. As microtubules guide the cellulose synthase complexes, this observed reorganisation pattern probably affects the cellulose deposition, contributing to the reinforcement of the cell wall in a particular position to cope with the extra-applied pressure. The age-effect is reasonable, since younger cells, which are actively shaping their cell walls, are more vulnerable to altered mechanical stresses while in leaves older than 19 DAS, the walls are more robust and therefore can sustain the applied forces.

Figure 1

Supracellular patterns. Two example images of a supracellular microtubule stress pattern (a). The continuous patterns visible on these images are marked in white lines on the images in b. The zoomed sections show circular supracellular patterns around stomata (marked by blue dotted line). (scale bar = 50 μm).

Figure 2

Time series of microtubule patterns. Image a shows the microtubule patterns in a leaf of 15 DAS at time point 0. Image b is taken at the same position but with a time interval of 3 hours and shows how microtubules were rearranged in parallel bundles within the cells. The zoomed sections show the altered microtubule patterns in more detail in the same cell at the initial time point (a) and after 3 hours (b). The supracellular patterns that appear after 3 hours are marked with a white line on image c. (scale bar = 50 μm). Image d presents the microtubule patters after 3 hours for a leaf of 22 DAS.

Figure 3

Time series of the microtubule response. Images of the microtubule patterns in a 15 DAS old leaf, after 0 (a), 60 (b), 120 (c) and 180 minutes (d).

Figure 4

Chambers with spacers in between microscopic slide and coverslip. The set-up is illustrated in image a. The root of the plant is pushed in the medium while the leaves are freely present in the upper space, which is filled with distilled water. The lower image is an illustration of the side view, showing that the leaves are not pressured between the microscopic slide and the coverslip. The microtubule response after 6.5 hours is shown in b for a leaf of 19 DAS.

Figure 5

Application of pressure at a specific location on the leaf. External pressure is exerted by a up-side-down placed glass petri dish (50 mm) (a). An 19 DAS old leaf can be divided in three distinct zones as shown on image b: zone of direct application of the pressure (grey), zone of microtubule response (blue) and the non-responding parts of the leaf (green). Representative images are shown in c for all three locations. (scale bar = 50 μm).

Figure 6

Stress patterns in the leaf. Image (a) shows the situation when a global external load is perceived by the stomata and generates a circular stress pattern. A rod-shaped application of external pressure (b) creates a perpendicular stress in the surrounding regions.