Table 1 Mean plant biomass 9 1 SE and mean concentration of macronutrients in leaves of the Zn–Cd resistant ecotype Plombie`res of S.
6 ulgaris grown for 5 weeks on orogenic soils in a greenhouse
a
Biomass mgplant Site
Macronutrients mmol g
− 1
d.m. Soil number
N P
K Ca
Mg 9.0 9 5.1
a
2400
de
70
d
11 918
c
Langelsheim I 363
c
115
a
12 Langelsheim II
10.5 9 1.7
a
2530
de
62
cd
745
b
382
cd
106
a
14 21.2 9 7.5
b
Welfesholz III 2100
c
78
e
1690
fg
534
ef
385
e
24.5 9 12.1
bc
1980
c
35
b
Welfesholz IV 1750
g
15 465
ed
378
e
Blankenrode 3
25.6 9 8.2
bc
1020
a
22
a
419
a
152
a
202
cd
25.9 9 8.4
bc
3950
g
39
b
1 1900
h
Wildemann I 606
f
182
c
28.0 9 8.5
bc
1730
b
39
b
Klosterrode II 1610
f
6 443
d
529
f
Welfesholz Ib 8
30.5 9 10.5
bc
2310
cd
40
b
1450
e
380
cd
521
f
35.2 9 13.2
c
2650
e
85
f
9 1040
cd
Marsberg II 305
b
128
ab
36.4 9 12.9
c
1650
b
69
d
Marsberg I 631
b
10 352
c
137
b
Plombie`res 16
41.5 9 18.3
cd
2230
cd
57
c
1180
d
293
b
693
g
42.6 9 14.0
cd
1800
bc
32
b
5 1190
d
Klosterrode I 428
d
355
e
59.9 9 22.9
de
2850
ef
73
d
Wildemann II 2350
I
2 393
cd
173
c
Welfesholz II 13
63.3 9 33.0
def
2540
de
82
ef
2320
I
410
d
356
e
95.8 9 28.4
fg
2170
c
87
f
1790
g
471
e
215
d
7 Welfesholz Ia
a
The plants are ranked according to biomass increase. Values with different superscripts indicate significant differences at least at PB0.05. The biomass data are from Ernst and Nelissen 1999.
leaf pairs after extraction with 80 vv aqueous acetone in the presence of small amounts of
quartz sand and Na
2
CO
3
and centrifugation. The absorption of the supernatant was measured at
470, 647 and 663 nm in a Pharmacia Ultraspec III. The concentration of chlorophyll a and b and
carotinoids were calculated using the equations given by Lichtenthaler 1987.
Anthocyanins were extracted by grinding the oldest leaves of 5 week-old plants with mortar
and pestle in the presence of quartz sand. The extraction medium was methanolHCl 991, vv.
After centrifugation, absorbance spectra of the supernatant were recorded with a Pharmacia Ul-
traspec III at 528 nm Kakegawa et al., 1991, which was the wavelength with the maximum
extinction. Cyanidinchloride was taken for cali- bration of the cyanidins.
2
.
4
. Phytochelatin analysis For the analysis of phytochelatins 3 – 5 pairs of
mature leaves were excised from two 5-week-old plants per pot, three pots per soil, and immedi-
ately frozen in liquid nitrogen. After homogeniza- tion with quartz sand and centrifugation at
27 000 × g for 20 min at 4°C the supernatant was analysed by a HPLC assay using post-column
derivatization or with 20 mM monobromobimane Sneller, 1999 modified after Rijstenbil and Wijn-
holds 1996. The samples were lyophilized and stored under vacuum until detection.
2
.
5
. Statistics Correlation between the various measured
parameters were calculated and tested for signifi- cance P B 0.05 by one way ANOVA. Multiple
comparison among means based on equal samples sizes were made by application of the T-method
Sokal and Rohlf, 1995.
3. Results
3
.
1
. Mineral elements
3
.
1
.
1
. Major nutrients Plants of this ecotype showed a high respon-
siveness to the soil conditions. The concentration
of the major nutrients in the leaves of plants grown on the various orogenic soils Table 1
varied the least for P with a factor of 4.0 from 22 to 87 mmol P g
− 1
dry mass and the most for K and Mg with a factor of 5.6 from 419 to 2350
m mol K g
− 1
and 6.5 from 106 to 690 mmol Mg g
− 1
, respectively; the concentration of N and Ca varied by a factor of 4.0 from 1020 to 3950 mmol
Table 2 Change of the mean metal concentration in leaves during growth from seedling to mature plants of the Zn- and Cd-resistant ecotype
Plombie`re of S. 6ulgaris on four orogenic soils
a
Plant mass Soil number
Metal concentration mmol g
− 1
dry mass Plant age days
Fe Mn
Zn Cu
Cd Pb
0.104
a
0.981
a
4.41
a
1.47
a
0.123
a
n.d.
b
0.63 9 0.15 mg 14
7 99.80 9 29.40 mg
3.15
b
35 1.37
a
2.64
b
0.243
b
0.008
b
B 0.010
c
228 0.013
b
0.99
a
0.003
c
1.73 9 0.28 g 0.115
c
2.25
b
1.51
a
14 0.84 9 0.34 mg
2.86
b
1.07
a
3.64
a
0.894
a
0.113
a
0.060
a
6 5.60 9 1.70 mg
6.62
c
2.59
b
35 7.50
b
1.390
b
0.016
b
0.220
b
228 1.19 9 0.28 g
0.72
a
4.47
c
6.30
b
0.495
c
0.001
c
0.240
b
3 14
2.660
a
0.266
a
0.44 9 0.21 mg 0.885
a
33.40
a
12.5
a
9.23
a
35 25.60 9 8.20 mg
13.80
b
12.5
b
16.50
b
0.130
b
0.025
b
0.810
b
0.58 9 0.29 mg 13.60
a
12 14.2
a
14 63.60
a
1.130
a
0.139
a
3.390
a
9.64 9 2.72 mg 21.90
b
35 11.4
a
143.00
b
2.590
b
0.056
b
1.720
b a
On the soil 3 and 12 the plants did not survive to maturity. Among each site and element data with different superscripts are significantly different at least at PB0.05.
b
n.d.; not determined. Table 3
Root mass and mineral element concentration in roots after 5 weeks of growth on orogenic soils in a greenhouse
a
Soil number Mineral element concentration mmol g
− 1
d.m. Root mass mg plant
-1
Zn Mn
Fe Mg
Cu Ca
P K
Cd 12.3
f
0.072
d
255
c
33
a
103
b
29
a
79
d
12 7.4
c
0.25 9 0.34 142.0
e
32.6
h
0.606
h
576
f
95
g
11 341
f
1.10 9 1.23 87
d
179
h
24.0
f
267.0
f
3.7
d
16 2.76 9 1.38
64
a
0.826
i
41
bc
152.0
e
229
d
39
b
124
g
27.1
f
0.062
cd
3.89 9 1.72 212
c
68
e
106
b
40
b
74
cd
18.9
e
8.2
b
13.4
f
9 0.283
g
4.02 9 1.62 583
f
45
c
190
d
91
d
99
f
23.9
f
24.0
c
1.9
c
1 6.6
e
22.3
c
4.5
b
42
b
0.076
d
134
e
15 228
d
58
d
380
de
4.16 9 2.99 22.6
g
0.070
d
434
e
63
d
14 332
f
4.20 9 1.62 207
f
157
h
6.6
bc
40.1
d
10 14.7
f
10.6
b
34.0
g
0.054
c
121
g
5.26 9 2.22 40
b
90
b
61
d
126
b
0.123
e
5.30 9 2.03 794
g
81
f
145
c
78
c
50
b
13.9
d
11.9
b
0.7
b
2 3.8
d
0.027
b
610
f
30
a
315
ef
210
f
23
a
2.6
a
13 8.8
b
5.41 9 3.11 33.4
d
12.9
f
6 5.48 9 3.26
0.110
e
320
d
14.2
d
33
ab
314
ef
255
g
78
d
0.009
a
8.02 9 2.86 433
e
29
a
287
e
204
f
65
c
7.6
c
5.2
a
15.2
f
8 13.2
f
0.188
f
305
c
39
b
5 288
e
9.09 9 2.64 206
f
40
b
11.3
d
22.2
c
0.2
a
0.140
e
105
b
30
a
59
a
34
ab
72
c
2.5
a
3 19.4
c
9.96 9 5.15 110
b
67
de
349
d
18.50 9 11.65 4.2
d
14.4
bc
5.3
b
87
e
0.064
cd
95
d
7
a
The mean root mass 9 1 SE is based on six plants, two per pot of each soil. The analysis of mineral elements is the mean of three samples, one per pot with two pooled plants. Data with different superscripts per element are significantly different at least
at PB0.05.
Table 4 Metal concentration in seeds of plants of the Cd–Zn-resistant
ecotype of S. 6ulgaris grown on orogenic soils
a
Element concentration mmol g
− 1
d.m. Soil number
Fe Mn
Zn Cu
1.65
f
2 0.83
c
1.70
e
0.13
ab
5 0.88
a
0.75
c
0.91
cd
0.22
c
0.93
d
6 0.79
b
0.87
a
0.17
b
0.67
bc
0.62
a
1.31
d
0.12
a
7 0.77
c
0.88
c
8 0.22
c
1.63
e
1.26
e
0.68
a
1.21
cd
0.43
d
9 1.04
bc
13 0.39
a
0.71
ab
0.10
a
0.61
b
1.22
e
1.21
cd
0.26
c
14 1.15
c
15 0.57
b
0.81
bc
0.15
b
2.07
f
16 2.50
g
2.62
f
0.13
ab a
Values with different superscripts are significantly different at PB0.05.
dons was quite different on the various soils already 14 days after emergence as shown by the selected
data set comprising plants with the highest soil 7 and lowest soil 12 biomass and with a medium
biomass, but dying prior soil 3 or surviving up to soil 6 seed ripeness Table 2. Levels of Zn were
the highest of all heavy metals; concentration below 5 mmol g
− 1
seedling dry mass allowed the finaliza- tion of the life-cycle. The regulation of the Zn level
was quite different on the various orogenic soils: it decreased significantly P B 0.01; soil 7 or doubled
P B 0.01; soil 6 without surpassing the obviously critical level of 10 mmol Zn g
− 1
leaf dry mass soil 3, 12 up to the end of the life-cycle. A decrease of
the Zn level in seedlings on soil 3 by nearly 50 within 3 weeks was insufficient for survival. Levels
of approximately 1 mmol Cu g
− 1
seedling did obviously not hamper the further development of
the plants. Lead concentration above approxi- mately 0.3 mmol g
− 1
seedling may have contributed to seedling mortality. In all seedlings, the Cd
concentration strongly P B 0.001 decreased be- tween 2 and 5 weeks after emergence.
Metal concentrations in roots are the result of uptake and translocation to the shoot. Root growth
was severely diminished Table 3 if the metal concentration in the roots was above approximately
20 mmol Zn, 5 mmol Cu g
− 1
andor 0.1 mmol Cd g
− 1
dry mass soil 11, 12, 16. N g
− 1
and from 152 to 606 mmol Ca g
− 1
. None of the nutrients in plant leaves, however, was
significantly P B 0.05 related to the biomass pro- duction within 5 weeks after emergence Table 1
or up to seed maturity Table 5. The concentration of major nutrients in roots Table 3 was also not
related to growth performance.
3
.
1
.
2
. Hea6y metals The metal concentration of hypocotyl and cotyle-
Table 5 Mean aboveground biomass 9 SE and the amount of heavy metals in the above ground biomass as percentage of the heavy metals
in the soil solution at the start of the experiment per pot
a
Soil number Above ground biomass g per pot
Metal amount in the above ground biomass as a of the amount in the soil solution
Fe Mn
Zn Cu
Cd Pb
1.60 9 1.13 93
19 14
19 11
4 2
6 8
10 4
1.66 9 0.47 21
19 10
2.14 9 0.92 195
3 16
10 13
2 4
1 2.43 9 1.05
323 82
15 6
8 13
6 3.58 9 0.85
126 94
125 17
4 12
3.98 9 0.77 119
14 15
32 5
1 7
524 4.20 9 0.82
13 3
8 29
77 2
5.19 9 0.84 100
7 7
17 3
0.1 6
240 64
5.65 9 0.95 438
5 5
2 36
70 17
5.58 9 1.68 26
9 15
4 9
10.90 9 1.02 126
13 52
36 7
1 11
a
The data are ranked according to increasing biomass.
Table 6 Concentration of chlorophylls Chl a, Chl b and cyanidin in the Zn- and Cd-resitant ecotype of S. 6ulgaris after 5 weeks of growth
on orogenic soils in a greenhouse
a
Chl a mg g
− 1
fresh wt. Soil number
Chl b mg
− 1
fresh wt. ratio Chl ab
cyanidin mmol g
− 1
fr.wt. Chlorotic
2 0.152 9 0.020
0.367 9 0.051 2.41
1.72 9 0.22 0.120 9 0.008
7 3.23
0.387 9 0.037 3.08 9 0.41
0.041 9 0.010 0.93
0.038 9 0.009 n.d.
b
11 0.046 9 0.008
12 0.048 9 0.009
0.96 n.d.
14 0.166 9 0.019
0.244 9 0.024 1.47
3.27 9 0.37 Chlorotic and rich in cyanidin
1 0.131 9 0.014
0.267 9 0.035 2.04
6.02 9 0.81 5
0.189 9 0.022 0.406 9 0.044
2.15 9.50 9 1.02
0.231 9 0.031 1.99
0.460 9 0.040 5.41 9 0.64
16 Rich in cyanidin
0.678 9 0.043 1.20
0.814 9 0.025 9.14 9 0.87
3 0.682 9 0.036
6 0.358 9 0.028
1.91 6.71 9 0.70
0.259 9 0.026 2.81
8.68 9 0.93 0.729 9 0.042
15 Green plants
8 1.070 9 0.098
0.284 9 0.026 3.77
3.37 9 0.45 0.294 9 0.034
1.92 0.565 9 0.041
2.31 9 0.12 9
0.724 9 0.055 13
0.217 9 0.030 3.34
2.66 9 0.23 10
0.662 9 0.043 0.325 9 0.021
2.04 2.94 9 0.42
a
Data are the mean 9 1 SE of three plants, one per pot, per soil.
b
n.d.; not determined.
Table 7 Mean concentration 9 1 SE of phytochelatins PC2, PC3 and heavy metals in mature leaves of S. 6ulgaris grown for 5 weeks on
orogenic soils in a greenhouse
a
Soil number PC3 nmol g
− 1
d.m. PC2 nmol g
− 1
d.m. Element concentration mmol g
− 1
d.m. Zn
Cu Cd
Pb B
2.0 5.5
d
1 0.16
ab
B 2.0
0.049
g
0.94
e
2 B
2.0 B
2.0 4.3
c
0.09
a
0.034
f
1.13
ef
B 2.0
16.5
f
0.13
a
B 2.0
0.025
e
3 0.81
e
4.4 9 42 5.3
cd
1.02
e
5 0.015
d
10.7 9 5.3 0.09
c
B 2.0
7.5
e
1.39
e
5.7 9 1.7 0.016
d
6 0.22
d
7 B
2.0 B
2.0 2.6
b
0.24
b
0.008
bc
B 0.01
a
11.8 9 15.2 1.6
a
2.13
g
18.1 9 4.5 0.001
a
8 B
0.01
a
12.5 9 15.2 2.7
b
3.36
h
9 0.031
ef
103.0 9 47.5 0.03
b
B 2.0
3.2
bc
0.35
c
B 2.0
0.005
b
13 0.05
b
B 2.0
7.0
e
3.00
h
14 0.010
c
6.6 9 0.2 0.27
d
B 2.0
4.2
c
0.60
d
B 2.0
0.016
d
15 0.09
c
B 2.0
102.5
g
0.38
c
16 0.820
h
B 2.0
0.29
d a
The detection limit of PCs was B2.0 nmol SH equivalents g
− 1
dry mass.
Fig. 2. Relationship among the Zn concentration in roots a, b and leaves c, d and the water-soluble b, d and total Zn concentration of the soil after 5 weeks of growth.
Fig. 2. Continued
With regard to Zn concentration in plants, there were two response ranges. The Zn concen-
tration in leaves and roots increased with increas- ing total soil Zn up to 220 mmol Zn g
− 1
dry soil Fig. 2a, c, but it was kept nearly constant if it
was related to the water-soluble Zn up to 150 nmol Zn g
− 1
dry soil in leaves Fig. 2b and 220 nmol water-soluble Zn g
− 1
in roots Fig. 2d. In this range the Zn concentration in roots remained
below 40 mmol, in leaves below 8 mmol g
− 1
dry mass. On its soil of origin 16, the Zn uptake
remained within this range, but the translocation into leaves resulted in very high Zn concentrations
above 100 mmol Zn g
− 1
dry mass. The regula- tion of Zn uptake by roots obviously failed on
extremely Zn-enriched soils 3, 11, 12. The Cu concentration of leaves increased more
or less linearly with the water-soluble and total Cu concentration of the soil, however, with a low
power of the regression function Fig. 3a, b. Only Cu in roots and the total soil Cu concentration
Fig. 3c, d had a significantly linear relationship P B 0.02. The concentration of Cd and Pb in
roots Fig. 4a, b were linearly related with the total soil concentration of Cd P B 0.01, r
2
= 0.52
and Pb P B 0.001, r
2
= 0.84. In the case of the
above-ground plant parts, the concentration of Cd, Fe, Mn, and Pb was not correlated with the
metal concentration in the soil solution. Enhanced metal exposure may affect the metal
loading of seeds and thus burden the next genera- tion. Although the metal content in seeds was low
compared to that in all other plant parts roots, leaves, stalks, calyx and capsules at the time of
seed maturity data not shown, the metal concen- trations of the seeds Table 4 varied by a factor
of 2.4 for Fe, 4.3 for Cu and Zn, 6.4 for Mn between the various orogenic soils. The concen-
tration of Cd and Pb in seeds remained below the detection limit of 0.001 and 0.01 mmol, respec-
tively. For two elements there was a linear rela- tionship of the seed metal concentration with that
of the soil: The Zn concentration of seeds signifi- cantly P B 0.001 increased with total and water-
soluble Zn in the soil; the Fe concentration was significantly P B 0.001 related to the total iron
level of the soil.
In our experiments with a defined soil mass and without leaching losses, availability of metals to
and uptake of metals by plants can be estimated by the ratio of the amount of metals in plants and
the amount of water-soluble metals in the experi- mental unit Table 5. From the water-soluble
amount of Cd and Pb less than 10 and 13, respectively, was accumulated in the above-
ground biomass. At maximum one third of water- soluble Cu was used by the plants, whereas the
use of the water-soluble Zn varied between 10 and 240; a value above 100 indicates that a replen-
ishment of the water-soluble fraction was neces- sary to keep up with the accumulation in the
plant. If plants took up more than 40 of the amount
of water-soluble
Zn, they
became chlorotic. From 3 to 94 of the water-soluble Mn
was present in the above-ground plant parts. In soils of the Cu mine from Marsberg 9, 10 the
amount of water-soluble Fe was so high that only ca. 20 was present in the above-ground plant
Fig. 3. Relationship among the Cu concentration in roots a, b and leaves c, d and the water-soluble and total Cu concentration of the soil after 5 weeks of growth.
Fig. 3. Continued
parts. In all other soils, the Fe amount in the plant was nearly equal to the water-soluble
amount in the pots 93 – 126 except on soils from the Zn mines at Plombie`res 16 and Wilde-
mann 1, 2 and the Cu mines from Klosterrode 5 where a replenishment up to a factor of 4 was
necessary to cover the plant demand.
3
.
2
. Cyanidins and chlorophylls Plants growing on zinc-enriched soils at Wilde-
mann 1, Plombie`res 16, and Blankenrode 3, and the Cu – Zn soils at Klosterrode 5, 6, and
Welfesholz 15 had dark-red leaves due to high concentrations of cyanidin varying from 5 to 10
m mol cyanidin g
− 1
fresh weight Table 6. Leaves of plants grown on several of these soils 1, 5, 16
were chlorotic in the upper part of the shoot and rich in cyanidin in the lower part of the shoot.
The concentration of cyanidin was negatively re- lated with the P concentration r
2
= 0.83, P B
0.001 and not significantly related with the leaf N concentration r
2
= 0.25, P \ 0.05.
After 5 weeks of growth, plants on the poly- metallic soils from Wildemann 1, 2, Klosterrode
5, Langelsheim 11, 12 and Welfesholz 7, 14
Fig. 4. Relationship among the metal concentration in roots and the total concentration of Pb a and Cd b after 5 weeks of growth.
and on the Zn-soil from Plombie`res 16 developed symptoms of chlorosis Table 6. The plants on the
soils with the highest metal concentration 11, 12 contained very low chlorophyll a and b and were
nearly yellow-whitish. They died prior to flowering. All other chlorotic plants except those on soil 5 were
delayed in flowering and had a low biomass produc- tion. The degree of chlorosis, i.e. the chlorophyll
concentration was negatively r
2
= 0.53, P B 0.01
correlated with the Zn concentration of the leaves. In non-chlorotic plants, the mean chlorophyll con-
centration varied from 0.56 to 0.81 mg g
− 1
fresh weight. One exception to this rule were plants with
very stunted growth on one of the Zn-enriched soils 3. The leaves had very high Zn concentrations
combined with the highest chlorophyll content of all investigated non-chlorotic plants 1.49 mg
chlorophyll g
− 1
fr. wt. versus 0.85-1.35 mg chloro- phyll g
− 1
fr. wt in normal green plants, Table 6.
3
.
3
. Phytochelatins Only leaves of plants grown on Cu mine soils 9
and on polymetallic soils with Cu concentration above 38 mmol Cu g
− 1
dry soil had phytochelatin PC2 levels above 2 nmol SH equivalents per g dry
mass Table 7. Plants with leaf Cu concentration above 1 mmol Cu g
− 1
dry mass contained also PC3 5, 6, 9. All plants on soils high in Zn, but low in
Cu had no detectable PC values. The Cd concentra- tion of the soils was obviously to low to induce PC
synthesis.
4. Discussion
