The Complexity of the Influence of Growth Substances, Heavy Metals, and Their Combination on the Volume Dynamics of Vacuoles Isolated from Red Beet (Beta vulgaris L.) Taproot Cells

Abstract

The plant vacuole is a very dynamic organelle that can occupy more than 90% of the cell volume and is essential to plant cell growth and development, the processes in which auxin (indole-3-acetic acid, IAA) is a central player. It was found that when IAA or FC (fusicoccin) was present in the control medium of vacuoles isolated from red beet taproots at a final concentration of 1 µM, it increased their volume to a level that was 26% or 36% higher than that observed in the control medium without growth regulators, respectively. In the presence of IAA and FC, the time after which most vacuoles ruptured was about 10 min longer for IAA than for FC. However, when cadmium (Cd) or lead (Pb) was present in the control medium at a final concentration of 100 µM, it increased the volume of the vacuoles by about 26% or 80% compared to the control, respectively. The time after which the vacuoles ruptured was similar for both metals. The combined effect of IAA and Pb on the volume of the vacuoles was comparable with that observed in the presence of Pb only, while for FC combined with Pb, it was additive. The use of IAA or FC together with Cd caused in both cases a decrease in the vacuole volumes by about 50%. The data presented in this study are discussed, taking into account the structure and function of the vacuolar membrane (tonoplast) and their changes in the presence of growth substances, heavy metals, and their combination.

Keywords: Beta vulgaris L.; FC (fusicoccin); IAA (indole-3-acetic acid); heavy metals; vacuole volume.

Figure 1

The effects of 1 μM indole-3-acetic acid (IAA) and fusicoccin (FC) on the volume changes of vacuoles isolated from red beet (Beta vulgaris L.) taproots. The vacuoles had been isolated directly onto glass slides by rinsing the surface of the fresh tissue slices with a control medium and a control medium with IAA or FC. The data points are the means of vacuole volumes (±SE) obtained from at least six single vacuoles. The volume of single vacuoles was calculated from the diameter of the vacuoles in a photographic image. The volume of the vacuoles was measured at the indicated times and converted to a percentage of the initial value (fixed as 100%). For the experiments, vacuoles with diameters in the range of 20–40 µm were selected. This figure also shows the volume changes of individual vacuoles that burst after a time longer than the vacuoles taken to calculate the mean values.

Figure 2

The photographs of the vacuoles incubated in the control medium and in the presence of IAA and FC. In order to show that the volume of the vacuole in the control medium practically does not change within 60 min, we showed a vacuole with a large diameter. The diameter of the vacuoles measured at 0 time is not the critical parameter that determines the time (at least over the first 60 min) after which the vacuoles disrupted.

Figure 3

The effects of 1 μM indole-3-acetic acid (IAA) or 1 μM fusicoccin (FC) applied together with Pb (100 µM) on the volume changes of vacuoles isolated from red beet (Beta vulgaris L.) taproots. The vacuoles had been isolated directly onto glass slides by rinsing the surface of the fresh tissue slices with a bathing medium. The data points are the means (±SE) from at least six independent measurements performed every 3 min for the diameter of a single vacuole.

Figure 4

The photographs of the vacuoles incubated in the control medium with Pb and in the presence of IAA or FC combined with Pb.

Figure 5

The effects of 1 μM indole-3-acetic acid (IAA) or 1 μM fusicoccin (FC) applied together with Cd (100 µM) on the volume changes of vacuoles isolated from red beet (Beta vulgaris L.) taproots. The vacuoles had been isolated directly onto glass slides by rinsing the surface of the fresh tissue slices with a bathing medium. The data points are the means (±SE) from at least six independent measurements performed every 3 min for the diameter of a single vacuole.

Figure 6

The photographs of the vacuoles incubated in the control medium with Cd and in the presence of growth substances (IAA and FC) combined with Cd.

Figure 7

A comparison of the effects of growth substances (IAA and FC), heavy metals (Pb and Cd), and their combination on the maximal volume changes of vacuole immediately prior to their rupture. Moreover, these changes are also shown as a function of time after which the vacuoles ruptured. Bars indicate means ± SE taken from Figure 1, Figure 3 and Figure 5.