Changes in membrane integrity uncovered with CLSM
We tested the functional integrity of Tetradesmus cells using the amphiphilic styryl dye FM 1-43 as a vital stain. This stain binds to lipids and therefore will only be seen along the plasma membrane of intact cells, whereas in damaged cells with a fragmented plasma membrane, the membranes of the organelles will be stained as well. Cells of the aquatic T. obliquus had intact membranes only when hydrated (Fig. 6a). In cells under osmotic stress only the plasma membrane was stained, but a heterogeneous pattern of staining was found (Fig. 6b). The most damage was done by rehydration: intense staining of intracellular material (Fig. 6c) suggests that this species is vulnerable to osmotic stress. By contrast, cells of the temperate soil alga T. dissociatus and the desert alga T. deserticola had only the plasma membrane stained (Fig. 6d-f and g-i respectively) in all three physiological states (hydrated, under osmotic stress, rehydrated), indicating preservation of membrane integrity through the osmotic stress-relief cycle.
An absence of changes in cellular volume after desiccation or plasmolysis in Tetradesmus species may be related to the fact that these algae lack a large central vacuole that is typical for many land plants and streptophyte green algae. In streptophyte plants, aquaporins located on the vacuolar and plasma membranes were shown to play a prominent role in desiccation tolerance (reviewed in Becker et al. 2020). The absence of a large vacuole may mean that even when fully hydrated cells of Tetradesmus do not possess a storage of free water, which can be lost. In this case, the definition of desiccation tolerance as the ability to survive 80–90% water loss (Oliver et al. 2000) may not be applicable to these algae. Moreover, it is impossible to determine cell water content of hydrated Tetradesmus cells due to their microscopic size; water creates a film outside the cells which cannot be removed without risking dehydration of the cells. Thus, the method of subtracting dry plant mass from the hydrated mass that is commonly used for land plants (e.g., Bartoškova et al. 1999, Proctor & Smirnoff 2000) cannot be used in unicellular algae. However, these algae exhibit the hallmarks of vegetative desiccation tolerance, as actively metabolizing cells can equilibrate with very dry air and recover from it upon rehydration.