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

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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
54 M. Volgger et al. fluorescence and transmission mode simultaneously to clarify the location of membranes within plasmolysed root hairs. Overviews of seedlings and whole roots were taken with a Coolpix 990 (Nikon) attached to a stereo microscope (Nikon). Results Root hairs are formed by bulging from root epidermal cells. They grow up to a length of 500 to 1,000 μm (Fig. 2a) in about 13 h. During bulge formation, growth is slow with 0.1 to 0.2 μm/min. At a length of 5 to 35 μm, growth speeds up continuously to 1 μm/min. This velocity is maintained until the middle of the total growth phase (about 7 h). Thereafter, growth continuously slows down till the end of the growth phase (Fig. 2 b, c); figures were taken in 10 min intervals, the light source was switched off between the takes. The resulting growth curve was confirmed by at least 100 random samples of root hairs that were not stressed by long-term microscopic observation. When root hairs cease elongation, the polar distribution is lost, and the vacuole progresses into the tip zone. The organelles distribute regularly over the whole length of the tube, and also large organelles reach up into the clear zone. The velocity of organelles, however, is maintained. Isotonic medium causes growth stop Up to a concentration of 150 mOsm mannitol, neither changes in growth nor cytoarchitecture of root hairs could Fig. 2 a–c Root hair elongation under normal growth conditions (n
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