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

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).