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Aluminium sensitivity of root cells related to aluminium internalization 4203 Root growth and detection of aluminium Effects of aluminium on root growth were observed on seedlings which, 2 d after germination (DAG), were transferred to Petri dishes containing agar-solidified nutrient medium with different aluminium concentrations (0, 10, 50, 100, 200, and 300 lM total concentration of Al in the form of AlCl 3 .6H 2 O). Root elongation was measured every day during a 7-d period. After 7 d of cultivation, aluminium was detected by staining whole roots with haematoxylin (Polle et al., 1978) or morin (Vitorello and Haug, 1997). Roots stained with haematoxylin were observed in bright field and morin fluorescence was visualized by an Olympus BX51 microscope (Olympus, Japan) equipped with BP 470–490 excitation filter, BA 515 IF barrier filter and a DM 505 dichroic mirror. Electrophysiology Measurements of the plasma membrane electrical potential difference (E m ) were carried out at 24 °C in root cells of intact Arabidopsis seedlings, 2 DAG, by the standard microelectrode technique as described by Pavlovkin et al. (1993). The perfusion solution contained 0.1 mM KCl, 1 mM Ca(NO 3 ) 2 , 1 mM NaH 2 PO 4 , 0.5 mM MgSO 4 ; pH was adjusted to 4.5. Diffusion potential (E D ) was determined by application of inhibitors (1 mM NaCN+1 mM SHAM) dissolved in perfusion solution. The influence of morin on E m was measured upon adding 100 lM morin to the perfusion solution. Effects of aluminium on E m were monitored during continual exchange of the perfusion solution by the experimental solution (50 lM AlCl 3 ) in perfusion solution, perfusion speed 5 ml min 1 ). After 5 min or 30 min of aluminium exposure the experimental solution was washed out with perfusion solution. Changes of E m induced by aluminium were measured continuously during the whole experiment. Insertion of the microelectrode into the cortical cells of the distal portion of the transition zone, located 150–300 lm behind the root tip, and of the proximal portion of the transition zone, located 300–400 lm behind the root tip (Verbelen et al., 2006), was performed under microscopic control. Measurements were evaluated separately for each zone. Live microscopy of aluminium internalization into the cells Arabidopsis seedlings 2 DAG were transferred to microscopic slides modified to micro-chambers by coverslips (Ovečka et al., 2005). The chambers were filled with liquid nutrient medium of the same composition as used for cultivation in Petri dishes, but without agar and placed into sterile glass cuvettes containing the same nutrient medium (pH 4.5). The seedlings were grown in a vertical position under light in the growth chamber. During a 12-h period the seedlings resumed stable root growth. Subsequently the micro-chambers were gently perfused with the aluminium-containing medium (50 lM AlCl 3 in nutrient solution, pH 4.5, perfusion speed 10 ll min 1 ). After a 30 min pulse treatment, aluminium was washed out and the roots of the Arabidopsis seedlings were stained with 100 lM morin for 20 min, using the same perfusion technique and then washed with the nutrient solution. After this exchange, internalization experiments were performed in the micro-chambers directly on the microscope stage during 3 h. For labelling endosomes and tonoplast, the styryl dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(8-(4- (diethylamino) phenyl)hexatrienyl)pyridinium dibromide) was used at a final concentration of 4 lM in nutrient solution, applied for 5 min prior to the aluminium treatment. Internalization of aluminium wasobservedinlivingrootsinrealtimebyaninvertedmicroscopeLeica DMIRE2,equippedwith theconfocal laser scanningsystemLeica TCS SP2 (Leica Microsystems Heidelberg, Germany). Excitation wavelength for FM4-64 was 514 nm and for morin 458 nm. Fluorescence was observed between 640 nm and 700 nm (FM4-64) and 480 nm and 510 nm (morin). FM4-64 internalization and BFA-induced compartment formation Working solution of 5 lM FM4-64 was prepared from the stock solution (1 mg ml 1 FM4-64 in DMSO). With FM4-64 the plants were incubated for 10 min and the dye was washed out before observation. Roots labelled for 5 min with the FM 4-64 were pretreated with BFA applied at a concentration of 35 lM for 25 min. The effect of aluminium was studied by the application of 90 lM AlCl 3 for 90 min before treatment with BFA and FM4-64. NO labelling and measurements in control and aluminium-treated root cells Detection of nitric oxide (NO) was performed by the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA; Calbiochem, USA). The roots were incubated with 15 lM DAF-2 DA for 30 min and washed before observation. As a negative control, they were treated with 10 lM of the NO-scavenger cPTIO (Lombardo et al., 2006). Examination was performed with a confocal laser scanning microscope system using standard filters and collection modalities for DAF-2 green fluorescence (excitation 495 nm; emission 515 nm). Fluorescence intensity was measured with the open source software Image-J (http://rsb.info.nih.gov/ij/). Results Influence of aluminium on root elongation and morphology Arabidopsis seedlings cultivated for 7 d on agar plates with different concentrations of AlCl 3 exhibited concentrationdependent inhibition of root growth (Fig. 1). Growth of the primary roots was only slightly reduced by 10 lM AlCl 3 (to 95% of the control values, not statistically significant according to t test at P¼0.05). Inhibition of root growth was statistically significant at 50 lM AlCl 3 as compared to control according to t test at P¼0.01. However, growth of the primary roots was reduced only to 89% of the control. The inhibition was more apparent at 100 lM and 200 lM AlCl 3 (59% and 45% of control growth, respectively, highly significant at P¼0.001), while root growth was fully inhibited Fig. 1. Root growth of Arabidopsis plants cultivated for 7 d on agar plates with different concentrations of AlCl 3 . Growth of the primary roots is progressively affected by 100 lM and 200 lM AlCl 3 while root elongation is inhibited completely at 300 lM AlCl 3 . Average of 20 seedlings per treatment (6SD).
4204 Illéš et al. Fig. 2. Effect of aluminium on elongation and morphology of the root system of Arabidopsis seedlings after 4 d cultivation. Position of root tips after transfer of seedlings to aluminium-containing agar plates is indicated by arrows. Inhibition of root elongation at 200 lM and 300 lM AlCl 3 is accompanied by enhanced formation of lateral roots. Representative of 20 seedlings per treatment. Fig. 3. Histochemical detection of aluminium in the roots of Arabidopsis after 7 d cultivation on agar plates by two different staining methods: haematoxylin staining (A) and morin staining (B). Haematoxylin stained aluminium strongly at 100 lM and higher concentrations of AlCl 3 while the reaction of morin to aluminium started to be strong at 50 lM AlCl 3 . Note the radial expansion of root cells at 100 lM and 200 lM AlCl 3 , but not at 300 lM AlCl 3 due to the complete inhibition of root growth. Representative of five seedlings per treatment. Bar¼100 lm. by 300 lM AlCl 3 (only 2% root elongation as compared to control plants, highly significant at P¼0.001; Figs 1, 2). Severe inhibition of root elongation was accompanied by radial expansion of the root cells at 100 lM and 200 lMAlCl 3 , but not at 300 lM AlCl 3 , due to the complete inhibition of root growth (Fig. 3). Root architecture of Arabidopsis seedlings exposed to lower concentrations of aluminium (10–100 lM AlCl 3 ) was almost unaffected. However, 200 lM and
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