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

Plasmolysis and cell wall deposition in root hairs 53
In brief, seeds were synchronised in distilled water for
2 days at 4°C and then germinated in Petri dishes on wet
filter paper for 1 day at 24°C under continuous light
conditions. Three- to 4-day-old seedlings were transferred
into micro-chambers between a cover slip and a glass slide.
Six layers of Parafilm “M” (Pechiney Plastic Packaging,
Chicago, IL, USA) acted as a spacer between slide and
cover slip. In vertical orientation, the seedlings grew in
sterile glass cuvettes for another 24–30 h under continuous
light. As culture medium, we used a phosphate buffer, pH
6.2, 25 mOsm. The medium just reached the open lower
edge of the micro-chambers allowing for free exchange of
the medium between chambers and the cuvette. During the
cultivation period, the seedlings were exposed to constant
light at 22°C. To prevent damage or unintentional changes
of growth, it was very important to protect the very
sensitive root hairs against mechanical stress. The custommade
growth chambers enabled us to take the roots to the
microscope stage and analyse them without further transfer
or manipulation. Prior to every experiment, the regular and
constant growth of the selected cells was controlled. To
assure standard conditions, we observed root hairs during
the most stable phase of growth at a length of 100–300 μm.
For adaptation studies of whole roots, the seedlings were
prepared as described above and grown under the respective
osmotic stress conditions for up to 30 h. Data were
taken at 24 h after transfer. Lengths of whole roots were
measured before and after transfer to the osmotic solution.
During the time of observation, wheat seedlings formed one
primary root and two lateral roots. Total root length was
calculated by the addition of the lengths of the primary root
plus lateral roots. Out of three seedlings per concentration,
a mean value was determined reflecting the increment.
Editing of data and graphic design was done in Microsoft
Access and Excel (Microsoft Office 2003). Statistics were
performed on SPSS[R] 10.0. Correlation between length of
root hairs and osmotic value of growth media was tested by
Spearman rank correlation for standard significance level of
[alpha]=0.01%.
Osmotic solutions
Osmotic stress of the plants was induced by exposure to
iso- and hypertonic solutions of glucose (Merck, Germany)
and D-mannitol (Merck, Germany) in concentrations of 100
to 650 mOsm. Under the microscope, the culture medium
of the micro-chamber was carefully removed with a strip of
filter paper and replaced by the osmotic solution with a
micropipette (chamber perfusion). For long-term experiments,
the buffer in the cuvette was replaced by the osmotic
solution. After germination and transfer to the microchambers,
the seedlings were grown in the osmotic medium
for up to 30 h. Osmotic values of solutions were measured in a
Micro-Osmometer (Model 3MO plus, Advanced Instruments,
Massachusetts, USA).
The osmotic value of root cells and root hairs was
determined microscopically and coincides with the state of
incipient plasmolysis (or limiting plasmolysis; Oparka 1994)
equalling the respective concentration of gradient osmotic
solutions.
Autofluorescence and fluorescent markers
The deposition of new cell wall material, mainly callose,
was visualised by its blue autofluorescence after UV
excitation. Selective staining of callose was performed by
chamber perfusion with 1% aniline blue at a concentration
in buffer or osmotic solution, respectively. After UV
excitation, aniline blue gave a yellow fluorescence.
The membrane potential dye DiOC 6 (3) (3,3′-dihexyloxacarbocyanine
iodide) (Molecular Probes, USA) was used
to stain the Hechtian strands, Hechtian reticulum and ER of
plasmolysed root hairs. DiOC 6 (3) was applied by perfusion
of the micro-chamber for 5 min, just prior to observation.
We used a concentration of 5 μg/ml dissolved in the
respective osmotic solution and rinsed off excess dye with
the same osmotic solution. DiOC 6 (3) has been successfully
used to stain Hechtian strands in onion and Tradescantia
cells (Oparka et al. 1994; Lang-Pauluzzi and Gunning
2000; Lang et al. 2004).
Alternatively, roots were exposed to a membrane selective
non-permeable styryl dye, FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium
dibromide)
(Molecular Probes). The dye has been widely used for
observing plasma membrane recycling (Betz et al. 1996;
Emans et al. 2002; Bolte et al. 2004; Ovečka et al. 2005). It
was applied by perfusion for 4 min right before observation.
We used a final concentration of 8 μM FM1-43 in buffer or
osmotic solution. With an excitation wavelength of 488 nm,
the emission maximum of FM1-43 lies at 600 nm.
Microscopy
A Labophot 2 (Nikon) with epi-fluorescence illumination
was used for conventional light microscopy. Autofluorescence
of cell wall components and aniline blue labelling
was detected after UV excitation in combination with a UV-
2A/DM400 (Nikon) emission filter set.
Furthermore, living root hairs were analysed by confocal
laser scanning microscopy (Leica DMIRE2) with an objective
×63, NA 1.32 (Leica). For DiOC 6 (3) and FM1-43 labelling,
the 488 nm excitation wavelength from an argon laser was
selected. Step sizes for z-series varied from 0.25 to 0.8 μm.
Stacks of images and maximum projections were generated
with the software associated to the laser scanning microscope
(Leica Confocal Software). Images were taken in the