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
Heavy metal-resistant ecotypes are mostly resis- tant to those heavy metals which are in surplus in
their natural environment Ernst et al., 1992. It was expected that the Cd- and Zn-resistant ecotype of
S. 6ulgaris will thrive well on all soils high in Cd and Zn with the exception of the soils with Zn and
Cd concentrations far above of that from the site of origin, i.e. Plombie`res cf. the Bronze Age
smelting sites 11 and 12. The death of seedlings 6 weeks after emergence on these latter soils Ernst
and Nelissen, 1999 confirmed this expectation in contrast to that on the Zn mine soil 3. On soil 3
the low pH of 3.9 enhanced the mobility of Cd, Pb and Zn and resulted in a very high uptake of these
heavy metals within the first 2 weeks after seedling emergence Table 2. Despite a very reasonable
biomass of 5-week-old seedlings and a decrease of the metal concentration, the internal concentrations
of Zn and Cd were obviously too high to ensure further growth.
Due to a lack of Cu-resistance of the Plombie`res ecotype Schat and Ten Bookum, 1992 and due to
the specificity of the various metal resistant mech- anisms Ernst et al., 1992, it was expected that the
Zn – Cd-resistant ecotype may have problems to perform well on Cu-enriched soils. But the biomass
production on Cu-enriched soils was equal to or surpassed that of the Plombie`res ecotype on its soil
of origin cf. Tables 1 and 3. This unexpected result may indicate beneficial metal interactions on poly-
metallic soils in contrast to the reported synergistic responses of a metal-sensitive ecotype of S. 6ulgaris
to high Zn and Cu as well as high Zn and Cd Sharma et al., 1999.
4
.
1
. Metal impact on roots The accumulation pattern of the various heavy
metals in roots showed that the Cd- and Zn-resistant ecotype of S. 6ulgaris was quite differently affected
by the water-soluble metal concentrations. In the case of Zn at mono-metal exposure, the
sequence of physiological injury started with an impairment of cell division after one to several days
Davies et al., 1991 resulting in a hampered root growth and root elongation in hydroponics under
controlled conditions Gregory and Bradshaw, 1965; Ernst, 1968. Such a strong reduction in root
elongation growth, although not metal-specific Gregory and Bradshaw, 1965, was registered in
seedlings on soils of the very metal-enriched Bronze Age smelting sites 11, 12 already a few days after
germination cf. Table 3. The diminished growth of the Plombie`res ecotype on its soil of origin may
be related to a not well-regulated Zn uptake com- pared to the root performance in all other orogenic
soils, perhaps due to an impairment of the low affinity Zn transporter Van der Zaal et al., 1999
andor the cation diffusion facilitator Paulsen and Saier Jr, 1997 by high Cd concentrations.
In contrast to Zn, the uptake of Cu can not regulated over a wide external concentration
range. At mono-metal exposure to Cu the most rapid physiological disturbance of non-adapted
ecotypes is that of the integrity of the root plasma membranes De Vos et al., 1989, 1991. It resulted
in K
+
-leakage already a few minutes after expo- sure as soon as the resistance limit to SH-reactive
metals Ag, As, Cu, Hg was surpassed Wain- wright and Woolhouse, 1977. As a result of this
process, the K concentration should be dimin- ished in metal-affected roots. The consequences of
this K
+
-leakage for the K status of the plants has never been measured in short-term exposure ex-
periments. In a medium-term hydroponic experi- ment with Cu-sensitive and Cu-resistant ecotypes
of S. nutans, the K status of roots and shoots, however, was not affected by exposure to increas-
ing Cu concentration Ernst, 1975. In soil-grown plants, K
+
-efflux from roots to the soil solution, however, cannot be directly measured due to the
generally high K
+
-concentration in soil solutions of more than 1 mmol l
− 1
Haby et al., 1990. In the experiments of this study, there was no signifi-
cant P B 0.05 relationship between the K con- centration and the Cu concentration of the roots
cf. Table 3 or the Cu concentration of the soil cf. Fig. 3a, b. Obviously, plants exposed for a
full life-cycle to enhanced Cu concentrations can repair the K
+
-leakage of the root plasma mem- brane. At multiple metal exposure, this impact of
Cu may be mitigated by a moderate surplus of Zn because Zn is important for membrane integrity
by protecting membrane lipids and proteins against
oxidative damage
of a
Cu excess
Marschner, 1995. The relatively good growth of the Zn – Cd resistant ecotype on moderately Cu-
enriched soils Ernst and Nelissen, 1999 may be a prove of this statement.
4
.
2
. Metal impact on shoots A damaged root will impair the ion uptake and
water economy, partially via the K metabolism of the
plant during
the further
development. Subtoxic to toxic metal concentration in seedlings
were already analysed prior or at the same time as the first chlorosis was visible. At a total and a
water-soluble Zn concentration of more than 200 m
mol and 0.5 mmol g
− 1
dry soil, respectively, the uptake and translocation of Zn to the shoots was
too high to prevent the plant from severe toxicity and finally death. Although the translocation of
metals from roots to shoots may be delayed in time Lolkema et al., 1984, it was sufficiently
rapid in S. 6ulgaris so that within a few days or weeks the metal concentration of stalks and leaves
was a good indicator of the metal exposure. The Zn- and Cd-resistant ecotype had a relatively high
threshold for Zn, behaving up to 200 mmol Zn g
− 1
dry soil as a shoot Zn excluder sensu Baker 1981 but perhaps better defined as Zn-regulator,
whereas the linear relation between plant and soil Cu was similar to that of a metal indicator sensu
Baker 1981.
Detoxification of metals in plant tissues and cells is necessary to ensure survival up to repro-
duction and seed production as the ultimate scope for the maintenance of a population. With the
exception of an enhanced Zn transport across the tonoplast in this Zn-resistant ecotype Chardon-
nens et al., 1999 most of the processes are related to metal transport via the xylem, i.e. preferential
accumulation of Cd and Zn in the leaf epidermis of S. 6ulgaris Chardonnens et al., 1998, precipi-
tation of heavy metals in cell walls of various ecotypes of S. 6ulgaris Ernst and Weinert, 1972;
Bringezu et al., 1999 and the metallophytes Thlaspi caerulescens Va´zquez et al., 1992 and
Minuartia 6erna Neumann et al., 1997, and in a few plant species decontamination by glandular
tissue Ernst, 1974; MacFarlane and Burchett, 1999.
4
.
3
. Metal impact on seeds In contrast with the vegetative plant parts, nu-
trient transport to the seeds is governed by the phloem. As a consequence, the metal concentra-
tion of seeds from plants growing on heavy metal- enriched soils was low compared to all vegetative
plant parts Table 4, confirming data from this and other plant species from heavy metal soils
Ernst, 1974. The positive relation between Fe and Zn concentration in the seeds with that of the
soil, however, may indicate that the seed con-
tributed to the metal loading of the young seedlings. Therefore the relatively high concentra-
tion of Cd, Cu, Pb and Zn in the seedling two weeks after emergence cf. Table 2 may be not
only the result of metal uptake and translocation to the cotyledons, but also the carry-over burden
from the field-collected seeds. How this burden will affect germination and seedling establishment
in the field is not known, but may explain the sometimes low germination percentage Ernst and
Nelissen, 1999.
4
.
4
. Physiological biomarkers
:
plant pigments The above-mentioned detoxification processes
may impede a direct relation between metal con- centration
and metal
toxicity. Physiological
biomarkers may be another reliable early warning system in plants Ernst and Peterson, 1994, as
long as they also predict plant performance up to reproduction. From the various metabolic pro-
cesses which are known to give a good correlation between short-term metal exposure and toxicity
for an overview see Ernst, 1998 most are studied in roots of plants growing in nutrient solution.
Visible changes of plant colours, i.e. increased anthocyanidin concentration and loss of chloro-
phyll chlorosis, may be a first indication of an insufficient detoxification of the metals resulting
in deregulation of a plant’s physiology. These symptoms are expected to be non-specific, not
necessarily related to a surplus Ernst, 1974, but also to a shortage of metals Khan et al., 1998.
4
.
4
.
1
. Chlorosis Chlorosis is a parameter of a disturbed
metabolism by hampering chlorophyll synthesis. In severely chlorotic plants of the Zn- and Cd-re-
sistant ecotype Plombie`res of S. 6ulgaris the chlorophyll content was decreased by more than
90 on soils with a surplus of many heavy metals, i.e. Cd, Cu, Pb and Zn 11, 12. A decrease in the
chlorophyll content was reported from various plant species grown on soils naturally enriched
with metals, especially Cu all over the world Reilly and Reilly, 1973; Ernst, 1974; Vardaka et
al., 1997. In the present study, low chlorophyll concentrations could not be related to Cu, but
partially to a surplus of Zn. This correlation may be indirect. A surplus of Zn can impede the Fe
uptake as shown earlier for the Zn-hyperaccumu- lator Cardaminopsis halleri and other Zn-resistant
plant species Ernst, 1996. One of the conditions causing chlorosis may be the low availability of
Fe in the soil solution. The water-soluble Fe concentration in soils 5 and 16 cf. Ernst and
Nelissen, 1999 and the Fe demand of the plants may indicate an imbalance resulting in Fe-chloro-
sis due to a too slow replenishment of Fe in the soil solution. Iron shortage of the soil will stimu-
late the roots of dicotyledonous plants to exudate protons and acidify the rhizosphere Marschner,
1995, thus enhancing the uptake of other heavy metals Ernst, 1996. Another aspect of chlorosis
may be a FeZn interaction causing cytosolic Fe shortage. The high Fe concentration in leaves of
plants grown on soil 11 and 12 showed that the chlorosis was not due to Fe-deficiency in the soil
Marschner, 1995. It may be the result of an insufficient cellular availability of reduced Fe in
the presence of a surplus of Zn or the competition of Zn for the Fe binding sites during heme synthe-
sis Van Assche et al. 1979. Plants with a medium degree of chlorosis on soils 1, 2, and 6 survived up
to seed maturity, but with increasing plant age the FeZn ratio decreased from 1.5 to 0.2. Thus the
replenishment of iron in the soil solution and the competition of iron with zinc in the cellular
metabolism
are two
completely independent
parameters thus reducing the indicative value of chlorosis.
4
.
4
.
2
. Cyanidin Anthocyanins can be formed as a reaction to a
lot of adverse environmental conditions. At nutri- ent shortage, such as N- and P-deficiency, a sur-
plus of
carbohydrates can
be stored
as anthocyanins Bergmann, 1983. If shortage is
caused by elements competing with the P-uptake system like As Macnair and Cumbes, 1987 dark-
red leaves appeared Otte and Ernst, 1994. In the case of S. 6ulgaris the anthocyanidin is cyanidin.
Its high concentration in leaves of plants on soils from Blankenrode 3 and Klosterrode 5, 6 were
caused by P-deficiency and to a lesser degree that of N deficiency. In every case high cyanidin con-
centrations are not indicating a surplus of heavy metals.
4
.
5
. Biochemical biomarkers
:
phytochelatins Another
sensitive indicator
of sublethal
metal exposure may be the level of phy- tochelatins PCs. The relationship between phy-
tochelatin synthesis and metal exposure was mainly tested in roots grown in solution culture
under controlled conditions Inouhe et al., 1994; De Knecht et al., 1995; Klapheck et al., 1995;
Meuwly et al., 1995; Keltjens and Van Beu- sichem, 1998; Sneller 1999. PCs were the result
of metal-imposed strain and were not related to the metal-resistance Schat and Kalff, 1992.
Therefore PC levels increased also in Cd- or Cu- resistant ecotypes as soon as the exposure level
exceeded the resistance level to that specific metal De Knecht et al., 1992. Only a few stud-
ies have analysed the PC level of metal-exposed plants in the field. Grill et al. 1988 found very
low PC levels in roots of Acer pseudoplatanus and S. 6ulgaris on a Zn mine, and the PC levels
in the current-year needles of Picea rubens af- fected by air pollution did not surpass 8 nM
Gawel et al., 1996. The present study with the Zn – Cd-resistant ecotype of S. 6ulgaris confi-
rmed the general picture of an initiation of PC synthesis by SH-reactive heavy metals Grill et
al., 1987: only plants exposed to high environ- mental concentrations of copper andor cad-
mium synthesized measurable concentrations of PCs being in good agreement with a high stimu-
lation of the PC synthase by Cd and Cu, and a low one by Zn Grill et al., 1987; Klapheck et
al., 1995.
Can the degree of metal exposure in the field be related to PC levels of leaves when the plants
have not evolved tolerance to environmentally high Cu andor Cd concentrations De Knecht
et al., 1995? The high concentration of PC2 and PC3 in mature leaves of plants grown on
the Cu soil from Marsberg 9, 10 did fit well with the suggested low Cu tolerance of the
Plombie`res ecotype Schat and Ten Bookum, 1992 and confirmed field studies on Betula pen-
dula and Brassica rapa grown on some of the soils of this study Ernst, 1999. But the high
PC-levels were not indicative for the perfor- mance of the plants during a full life-cycle be-
cause plants with high PC-levels were able to survive and produce a good seedlot. Therefore
PCs may be indicative for a transient distur- bance of the metabolism with some protection
of metal-sensitive enzymes Kneer and Zenk, 1992, but they do not allow a prediction on the
performance of the plant up to reproduction.
5. Conclusions
