Stress treatments and inhibitor treatments
To impose UV-C stress, the leaf discs were irradiated for 2 min from a
distance of 12.5 cm by a linear fluorescent bulb (λmax =
254 nm, 15 W, Germicidal, General Electric, Japan). To impose aluminium
stress, the cells were collected and resuspended in the medium described
above (3% sucrose, 3 mM CaCl2, pH 4.5) before adding
AlCl3 (Sigma, Deisenhofen) to a concentration of 200 μM
(Ahad & Nick, 2007). In case of leaf discs, the treatment was conducted
in petri dishes with AlCl3 freshly dissolved in
distilled water (1 % w/v). To assess the role of actin filaments,
Latrunculin B as inhibitor of actin polymerisation, and phalloidin, a
compound stabilising F-actin (both Sigma, Deisenhofen, Germany) were
used in a final concentration of 1 μM diluted from an ethanolic stock
solution.. Diphenylene-iodonium chloride (DPI, Sigma-Aldrich,
Deisenhofen, Germany), diluted to a final concentration of 20 μM from a
stock in DMSO was used to inhibit the plasma membrane based NADPH
oxidase. To examine the influence of MAPK signaling, the inhibitor
PD98059 targeted to mitogen-activated protein kinase kinases (MAPKKs)
(Sigma-Aldrich, Deisenhofen, Germany), was dissolved in DMSO and used in
a final concentration of 50 µM. All treatments were accompanied by
solvent controls, where the maximal concentration of solvent (never
exceeding 0.1% v/v) used in the test samples was administered. If not
stated otherwise, the treatments with aluminium or the inhibitors lasted
for 2 hours. All experiments were performed at day 4 after
sub-cultivation, when the culture had completed proliferation and was
undergoing cell expansion. For the experiments with leaf discs, fresh,
fully expanded leaves (plastochrones 4 and 5) of uniform size were used.