A comparative structural analysis of direct and indirect shoot ...

56 M. Volgger et al.
The plasma membrane remains connected to the cell wall
after plasmolysis
During strong plasmolysis (350 mOsm and above),
Hechtian strands are formed (Fig. 5). DiOC 6 (3), a
membrane-selective dye, was used to visualise the thin
Hechtian strands and the Hechtian network close to the
cell wall. Figure 5a, b shows this membranous network at
different focal planes. It is evident that there is no
symmetric pattern of the strands. The Hechtian strands
are anchored to the shank of the root hair and also reach
towards the tip (Fig. 5 a, b, d). In bright field mode, the
Hechtian strands are merely visible (Fig. 5c). Alternatively
to DiOC 6 (3), these thin strands are brightly stained with
FM1-43, a membrane-selective styryl dye (Fig. 5d). FM1-
43 is only fluorescent when incorporated into the plasma
membrane and has been widely used as endocytosis
marker (Emans et al. 2002; Bolte et al. 2004; Ovečka
et al. 2005). With FM1-43, we observed Hechtian strands
emerging from the main protoplast as well as from subprotoplasts.
The strands are anchored to the flanks of the
root hair wall, as described for DiOC 6 (3), and emerged
also from the clear zone at the very tip of the root hair
(Fig. 5d). Thus, strong attachment sites for Hechtian
strands have to be incorporated already within the very
young, newly deposited cell wall.
Root hairs adapt to osmotic stress
Fig. 4 a–c Root hair tips under the fluorescence microscope. a–c Cell
wall deposition at 300 mOsm mannitol; a callose staining with aniline
blue (arrow). b callose/cellulose autofluorescence after UV excitation
in root hair tip showing dispersed material and a bright band of newly
formed cell wall (arrow). c Superimposing autofluorescence of wall
material after UV excitation and brightfield shows the plasmolysed
protoplast. Asterisks mark the plasmolysed protoplast. Bar 10 μm
cell wall that had been built at 400 mOsm becomes clearly
visible in the emptying plasmolytic space (Fig. 5f).
The intracellular organisation of the cytoplasm is maintained
at first. Only after 2 h in strong hypertonic solutions
over 350 mOsm, we observed a reorganisation of the
cytoarchitecture: the reverse fountain streaming is changed
into circulation streaming, and the clear zone at the tip is
reduced, however maintained. The position of the nucleus as
well as the velocity of organelle movement is constant.
In another set of experiments, we wanted to test the
adaptation of root hairs to osmotic stress conditions. For
that purpose, wheat roots were grown for 24 h in glass
cuvettes containing the osmotic solutions with concentrations
of 150 to 450 mOsm. Growth of root hairs and the
ability to form new root hairs under long-term osmotic
stress were tested as well as the development of whole
roots.
Figure 6 shows a significant reduction of the total
number of root hairs built (Fig. 6 a–d, dark field) and length
of root hairs (Fig. 6e–h). Under control conditions and up to
isotonic 150 mOsm, root hair development appears normal
(Fig. 6a, e). At 250 mOsm, less and shorter root hairs are
built than at lower concentrations (Fig. 6b, f). Interestingly,
even after 24 h, newly formed root hairs appear in osmotic
solutions of 350 and 450 mOsm mannitol, when normal cells
react with strong plasmolysis. Under these long-term osmotic
conditions, root hairs do not grow beyond a length of 50 to
100 μm, but their cellular organisation is the same as in
control hairs (Fig. 6e–g). Only at 450 mOsm, the clear zone
at the tip is not developed properly (Fig. 6h). Plasmolysis of
these “hardened” root hairs is only possible with concentrations
that are 200 mOsm stronger than during cultivation
(data not shown). Even so, they show only mild plasmolysis