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.