2. Materials and methods
2
.
1
. Plant material N. tabacum L. cv. Samsun NN plants were
grown in soil in a greenhouse at 18 – 22°C and 40 – 60 relative humidity. In vitro plants were
cultured in Murashige and Skoog [11] medium without growth regulators and with 2 sucrose
under a light – dark regime of 16:8 h at 3000 lux and a mean temperature of 24°C.
The cell suspension line 425 was started from callus of S275N, a suspension-cultured line of
cloned N. tabacum L. cv. Havana 425 pith tissue [12,13], which was kindly provided by F. Meins
Basel, Switzerland. The culture was initiated and maintained as described previously [14].
2
.
2
. Induction of CBP
20
Greenhouse plants used for induction of CBP20 were 12–14 weeks old 3 weeks before flow-
ering. At this stage, they had 16–18 leaves. In vitro plants taken for the same kind of experi-
ments were, if not otherwise noted, 4 – 6 weeks old with 8 – 10 leaves. Leaves were derived from ma-
ture source to sink leaves as described in the figure legends. For wounding leaves, shoots and roots
were scratched with razor blades arranged with a distance of 10 mm and subsequently placed in
dishes in distilled water. The dishes were gently shaken on a gyratory shaker 60 rpm in the
dark at room temperature for different times as indicated.
For salicylic acid treatment, in vitro plants were sprayed with a solution of 50 mM and, after 48 h
fully expanded leaves were harvested and frozen in liquid nitrogen.
Suspension-cultured cells
were filtered
and washed as described by Kunze et al. [14] and
subsequently transferred into media containing different heavy metals in different concentrations
as indicated in results. After incubation for differ- ent times, cells were harvested by filtration and
extracted as described earlier [15].Each experiment was repeated at least twice.
2
.
3
. Transcription inhibition experiments Floating experiments with leaf disks were per-
formed as described by Jacobs et al. [16] using actinomycin D Sigma, Germany at 100 mg ml
− 1
. After incubation with and without actinomycin D
for 2 and 4 h, leaves were used for RNA extrac- tion as described below.
2
.
4
. RNA extraction and Northern blot experiments
Total RNA was prepared as described by Loge- mann et al. [17], separation of RNA in denaturing
agarose gels, blotting and hybridization of nylon membranes were performed as described by Her-
bers et al. [18]. The CBP20-containing fragment isolated by the Qiagen kit Qiagen, Germany
from plasmid DNA of E. coli was used as probe.
2
.
5
. Protein extraction and immunoblotting Cells were collected on 56-mm pore-size nylon
filters from suspension cultures after different in- cubation periods as indicated in figure legends.
Extracts of cells, leaves, shoot segments and roots were prepared by freezing the material in liquid
nitrogen, grinding with a mortar and pestle, and vortexing the ground material with five glass beads
for 3 min in 1 ml buffer TNT 500 mM NaCl, 0.02 Triton X-100, 50 mM Tris-HCl, pH 8.0 per
1 g fresh weight of cells. After centrifugation 34,000 x g for 15 min the supernatant was used
to measure the protein concentration by the method of Bradford [19] using bovine serum albu-
min BSA as a standard. Five mg protein per lane were separated by SDS-PAGE as described by
Laemmli [20] except for using gradient gels from 11 to 20 polyacrylamide. Immunoblot analysis
was performed as described elsewhere [21]. The antibody directed against CBP20 was prepared as
described by Hensel et al. [15]. The antibody raised against the large subunit of the
D
-ribulose- 1,5-bisphosphate carboxylase rubisco was kindly
provided by
K.-H. Su¨ß
IPK, Gatersleben,
Germany.
2
.
6
. Chlorophyll assay Chlorophyll contents were determined as de-
scribed by Hanfrey et al. [22]. For this leaf, discs were excised in triplicate from different areas of
each leaf. Four parallel samples were used to calculate the standard deviation using the com-
puter program ‘Excel 5.0’.
3. Results and discussion
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