Sequestration at the cell wall and plasma membrane facilitates zinc tolerance in the moss Pohlia drummondii

Bryophytes are, evolutionarily, between the green algae and flowering plants that lack proper vascular and root systems as well as a protecting cuticle on the gametophyte. Bryophytes are sensitive to environmental stress factors and are widely used as bioindicators. However, bryophytes also show a high physiological flexibility and are able to inhabit ecological niches that range from highly irradiated peat bogs to salt lakes and metal-contaminated sites. The moss Pohlia drummondii lives on metal-contaminated habitats in Austria that contain high amounts of copper, iron and zinc. Zinc and copper are essential micronutrients for plants but are harmful if they are applied at high doses. Our experiments showed that P. drummondii prospered in vitro on solid media spiked with 1mM ZnEDTA and 10mM ZnEDTA. Using energy dispersive X-ray microanalysis (EDX), we detected the uptake and accumulation of zinc from the medium into the gametophyte, but the cellular location of the metal remained unclear. After breaking open the cells in liquid nitrogen, we tried to differentiate between the cell wall and the inside of the vacuole, which were both putative sites for heavy metal sequestration. However, we could not detect a significant difference between the two fractions. The zinc-specific fluorescent dye FluoZin™-3, which was applied to plant cells for the first time, produced more satisfying results. The cell walls of P. drummondii grown on both zinc concentrations showed bright fluorescent labelling, whereas the control cells showed no staining. Plasmolysis of the zinc-treated cells indicated that the dye entered the preplasmolytic space but did not pass the plasma membrane of the leaf cells. From these results, we propose that the cell walls of P. drummondii together with the plasma membrane formed a functional barrier to protect the cytoplasm from harmful doses of zinc, thereby providing an effective mechanism for P. drummondii to tolerate metal contamination and survive on zinc-loaded soils.