Environmental and Experimental Botany, Vol. 33, No. 4, pp. 575 589, 1993 0098 8472/93 $6.00+0.

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Printed in Great Britain. (Q 1993 Pergamon Press Ltd

M I N E R A L S T A T U S OF D I C O T Y L E D O N O U S C R O P PLANTS IN
R E L A T I O N T O T H E I R C O N S T I T U T I O N A L T O L E R A N C E TO LEAD

D A N U T A MARIA ANTOSIEWICZ
Laboratory of Electron Microscopy, Institute of Botany, University of Warsaw,
Krakowskie Przedmiescie 26/28, 00-927 Warsaw, Poland

(Received 1 May 1992; accepted in revisedf~rm 4 January 1993)

ANTOS~EWmZ D. M. Mineral status of dicotyledonous crop plants in relation to their constitutional tolerance
to lead. ENVIRONMENTALAND EXPERIMENTALBOTANY 33, 575 589, 1993. Four crop species
mustard, flax, tomato and sunflower used in experiments, were grown in the growth chamber
in solution culture. The plants were used in the following studies: (i) constitutional tolerance to
Pb (index of tolerance was estimated using Wilkin's root test with 0, 5, 10 and 20 mg dm :t Pb
concentrations in Knop's medium deficient in PO.;- :¢ ions); (ii) tolerance to deficit of P, Ca and
Mg and to general nutrient stress (50 x diluted Knop's medium) as determined by dry matter
yield; and (iii) effect of Pb and used nutrient regimes on mineral status of the plants, by estimation
of internal ion concentration. The species most tolerant to Ph was tomato. Tomato was also the
least tolerant to P deficit (although the test for Pb tolerance was conducted in medium without
P()~ :~ions). The least Pb-tolerant species, mustard and sunflower, were moderately tolerant or
non-tolerant to the mineral deficiencies used in the study. High constitutional tolerance to Pb
in tomato was associated with the highest tissue level of Ca during administration of Pb, and
with the highest tolerance to Ca deficit. The magnitude of the Pb concentration in the media
and its absolute amounts in the roots and shoots were not proportional to the degree of Pb
tolerance. Two of the least Pb-tolerant species had the highest (mustard) and lowest (sunflower)
root Pb concentrations. The absolute amount of Pb in the roots was highest in mustard. However,
the amount of Ph which was transported to the shoot in relation to the total amount of Pb taken
up by the plant was the highest tbr the least tolerant species, sunflower (43.4~o).

Key words: Constitutional Pb tolerance; Mg-, Ca-, P-starvation, internal mineral concentration.

INTRODUCTION find w i t h i n one species, p o p u l a t i o n s i n h a b i t i n g

T h E toxic effects of Pb (as well as o f o t h e r m e t a l s
p o l l u t i n g the e n v i r o n m e n t such as Zn, Cd, C u or
AI) c a n i m p o s e on plants a s t r o n g n a t u r a l selec-
tion tbr t o l e r a n c e , ~''3~''39i l e a d i n g to the devel-
o p m e n t o f p o l l u t a n t - t o l e r a n t p o p u l a t i o n s . Since
this t y p e o f t o l e r a n c e d e v e l o p s in response to the
presence o f m e t a l s in the e n v i r o n m e n t , it is t e r m e d
i n d u c i b l e t o l e r a n c e . ~5~6~ S u c h p o p u l a t i o n s are
often simply d e s c r i b e d as t o l e r a n t , w i t h o u t speci-
t}¢ing i n d u c i b l e n a t u r e . T h e r e f o r e , it is possible to
575
metal-enriched habitats exhibiting inducible
t o l e r a n c e w i t h respect to a specific m e t a l , a n d un-
p o l l u t e d " n o r m a l " p o p u l a t i o n s i n h a b i t i n g un-
p o l l u t e d sites a n d defined as n o n - t o l e r a n t
p o p u l a t i o n s . 2'5"a'39 W h e n the d e g r e e of t o l e r a n c e
of v a r i o u s species o f plants f r o m u n p o l l u t e d areas
was studied, it was f o u n d t h a t these species can
differ w i d e l y in their t o l e r a n c e thresholds.' r.~.2~ A
species was t b u n d w h i c h h a d such a h i g h level
o f i n h e r e n t t o l e r a n c e t h a t it was able to col-
onize m e t a l - c o n t a m i n a t e d e n v i r o m n e n t s w i t h o u t
576 D . M . ANTOSIEWICZ
evolving a tolerant race. TM In spite of certain
reservations, > it has been accepted that
plants may possess constitutional tolerance to
metals. ~:''<:~'4> YVide-scale studies have not yet
explained tile difference(s) between the mech-
amsms of both types of tolerance. The
genetic basis of inducible tolerance is un-
questionable, ~,28.42: whereas in the case of con-
stitutional tolerance, its magnitude may, for
instance, be determined by the plant's ability to
adapt existing environmental conditions within
tile limits of its own genotype. It is thus possible
that the "physiotype ''~ which thshions the
physiological characteristics of a given plant or
group of plants, such as the generally high or low"
level of ion take-up fi'om the environment, the
ability to store ions, etc., may determine the
plant's ability to adapt to, say, an increased level
of Pb. Physiotype may affect the degree of con-
stitutional tolerance to metal(s).
It is known that the unt~tvorable effect of metals
on a plant is manifested, fbr example, by inhi-
biting the normal uptake and utilization of min-
eral nutrients. 1°'1'-''1~:1'4°~It has been found that
plants from a population tolerant to a metal can
protect themselves fi'om this type ofadverse eft~ct
by developing resistance to deficits of those
macroelements whose uptake and translocation
are affected by the metal in question/ul<lTl!~' This
type ofdet~nse against harmful effects of metals is
characteristic of plants fi'om tolerant populations
(with inducible tolerance to metals). Their tol-
erance to the metal is associated with an acquired
change in mineral metabolism; this type ofdet~nse
is lacking in plants fi'om normal, non-tolerant
populations. It cannot he excluded that for plants
fiom unpolluted hahitats, the degree of con-
stitutional tolerance to a metal may be dependent
on fhctors of their "physiotype" such as an
inherent resistance to deficits of those macro-
elements whose metabolism is afi>cted by the
metal and the mineral status of the plant.
For the present study, it was hypothesized that
the degree of constitutional tolerance of a plant
to Pb may be associated with such traits as toler-
ance to deficits of'specific macroelements and the
plant's ion status. Ca, Mg and P intra-plant status
were chosen for the following reasons. (1) There
are reports suggesting that Ca, M g and P play a
role in the tolerance of plants to otherwise toxic
effects of various metals such as A1, Zn, Cd or
Pb.'l'~'2°':~°':~:~4':~(2) Administration of Pb causes
a decline in tissue calcium and magnesium
levels. ~1<1~'21'¢°'41~ (3) Inclusion of P in this in-
vestigation was considered important because
according t o Z I M D A H L ~'44' and MALONE el al., ~29~Pb
in a plant is deposited in the form of P compounds;
therelbre, P metabolism may be important in the
mechanism of tolerance to Pb. It should be added
that tests determining tolerance to Pb are con-
dueted in the absence ot'PO4 :~ions in the medium
due to the precipitation of Pb phosphate. ~3
The ot~jective of" this study was to determine if
increased constitutional tolerance of plants to Pb
is related primarily to their Ca status or to the
degree of tolerance to Ca deficit. Mg and P status,
resistance to Mg- and P-deficits and general nutri-
ent stress were also analyzed.
MATERIALS AND M E T H O D S
The experiments involved plants obtained from
unpolluted agricultural habitats. Four species
diftering in their nutritional requirements were
chosen, based on descriptions given by HERSE: ~3
Sinapis alba L. (mustard) and lycopersicon escu-
lentum Radek (tomato) (i.e. plants with high min-
eral requirements), Linum usilalissimum L. (flax)
(a plant with generally low nutritional require-
ments), and ttelianlhus annuus Sungold (sunflower)
(a plant with a moderate nutritional require-
ment). The tbllowing experimental topics were
studied: (i) constitutional tolerance to Pb; (ii)
tolerance to Ca-, Mg- and P-deficit and to general
nutrient stress; and (iii) mineral status of the
plants.
Seeds were germinated in darkness in Petri
dishes on filter paper moistened with distilled water
or Knop's medium. Seedlings with 1.5 2.3-cm-
long roots were transferred to plastic containers,
continuously aerated and cultivated as described
below. K n o p ' s medium, prepared from distilled
water, was the basic medium. Culture conditions
were: growth chamber, 23/19°C day/night, 16 hr
photoperiod and q u a n t u m flux density (PAR)
250 #mol m 2 s-i provided by Polam fluorescent
tubes Flora. The plants were collected after an
appropriate growth period (see below). The roots
were washed in distilled water. Alter separating
the roots fi'om the above-ground plant parts, their
 MINERAL STATUS OF CROP PLANTS AND Pb TOLERANCE
t)esh weights were determined. T h e y were then
dried at 90°C and their dry weights determined.
Mineralization of samples and determination of"
Ca, Mg, P and Pb concentrations were done
according t o NAPEK. :33 Samples were wet-ashed
using a mixture of concentrated HNO:~, HC104
and H~SO 4 ( 8 : 2 : 1 ) . Ca, Mg and Pb concen-
trations were determined using a flame atomic
absorption spectrophotometer (PYE U N I C A M
SP-900) against standard solutions (Merck).
P-content was determined by the vanado-
molybdophosphate method using Autoanalyzer
AA II, T E C H N I C O N . Statistical significance
was evaluated using Student's/-test. (2'~'
P-, Ca- and Mg-starvation experiments
Seeds were germinated in distilled water and
plants grown in 6.5-1 plastic containers for 21
days. T h e plants' tissue Ca, Mg and P contents
were determined as described above. The med-
ium was changed once on day 10. T h e experiment
was conducted in triplicate with 20 plants in each
replicate, i.e., [br each experimental regimen 60
phmts were used. T h e regimens were: (i) control
medium; (ii) medium diluted 50 x (1 pt med-
i u m + 4 9 pt H~O); (iii) medium lacking P; (iv)
medium lacking Ca; and (v) medium lacking Mg.
Rool lest
The degree of tolerance to Pb was determined
by the method described by WILKINS.: ~ T h e roots
growing in medium with or without the metal
were measured on days 1, 3, 5 and 7 of the experi-
ment. A "tolerance index" (7"1) was calculated
as a percentage value from the ratio
rill_
root growth increment in + Pb treatment
x 100.
root growth increment in control ( - Pb)
Pb was administered in the fbrm of PbC12 in
three concentrations: 5, 10 and 20 mg d m 3 in
K n o p ' s medium without phosphate ions (because
of Pb precipitation in the presence of p O4- 3 ions)
The seeds were germinated in K n o p ' s medium.
The plants were grown in 2.5-1 plastic containers
fbr 7 days. After 7 days the plants were collected
and their Ca, Mg, P and Pb contents determined
by the method described above. T h e experiments
were conducted in triplicate with 15 plants in
577
each replicate, i.e., for each regimen 45 plants
were used.
RESULTS
The mineral composition of control plants
Changes in the levels of the studied elements
in plants growing under various conditions are
presented as relative values (i.e. percentage of
control values) in Table 1. T w o control media
were used in this study: (i) K n o p ' s medium lack-
ing P was used in the 7-day experiments with Pb,
and (ii) K n o p ' s complete medium in the 21-day
experiments to determine the tolerance of plants
to mineral stress.
Experiments with continuous administration of Pb
Root test. T o m a t o (Iycopersicon) had the highest
index of tolerance ( T I) for all tested con-
cemrations (Fig. 1). The remaining three species
were distinctly less tolerant to Pb, with flax being
second after tomato. Sunflower and mustard had
the lowest 771. After 7 days at the highest con-
centration of Pb, 20 mg d m ~, the TI was found
to have fallen to zero tbr three species, while that
[br the most tolerant species, tomato, was ~ 30cli~.
Thus, in terms of TI, tomato > flax >
sunflower = mustard.
Dry matter yield. Only mustard (Sinapis) exhi-
bited a statistically significant decrease in shoot
dry matter after 7 days of growth in the presence
of 20 mg dm :~Pb (Fig. 2). For the other species,
drv matter yield did not differ significantly from
control values (Fig. 2). However, the root dry
matter yield of mustard, flax and sunflower
decreased under the influence of Pb; the smallest
decrease was in flax, the second most Pb-toleranl
species studied. T o m a t o was most tolerant to Pb;
its root dry matter yield significantly exceeded
that of the controls at all tested Pb concentrations.
Uptake of Pb. The Pb taken up by the plants
accumulated mainly in the roots (Table 2). The
difl'erences between the root and shoot Pb con-
centrations were very large, greatest in the
tomato, a 43.3-tbld difference between root and
shoot (at a Pb concentration of 10 mg dm :~ in
the medium, Pb concentration in the root was
25.200 mg k g J dry wt, while in the shoot, 580.9
mg kg 1). The smallest difference (8-tbld) was
tound in sunflower (at 20 mg Pb dm t in the
578 D . M . ANTOSIEWICZ

Table 1. Concentration qf P, Ca and Mg (%) and dry maUeryield per 1 plant (rag) in the control plants after 7 @ s oJ
cultivation ( Knop'.~ .wlution without P) and qfier 2t da> of cultivation ( Knop's solution)

Days Shoot Root

o1
growth P Ca Mg Yield P Ca Mg Yield

Sin@is
7 0.88_+0.09 1.20_+0.05 0.67±0.06 8.6_+0.6 0.84-+0.03 0.76+0. t4 0.36-1-0.05 1.6_+0.1
21 0.97_+0.07 1.96±0.21 0.51 _+0.05 48.0±6.0 2.91 -+0.40 1.69±0.38 0.25±0.03 7.3_+0.5
Linum
7 1.00-+0.11 0.71±0.13 0.7l±0.07 4.1_+0.5 0.59=0.06 0.49_+0.03 0.84_+0.08 1.1±0.1
21 1.30±0.09 1.60±0.20 (I.51±0.02 13.8_+1.2 2.07_+0.20 1.08±0.10 0.56_+0.05 3.5_+0.5
Lycopersicon
7 0.72±0.01 1.24_+0.1l 0.86-+0.08 3.8_+0.2 0.56-+0.10 1.17±0.07 0.60_+0.10 0.6--+0.1
21 0.81-+0.07 1.93_+0.21 0.52±0.07 49.0_+9.1 1.99_+0.38 1.01±0.11 0.46_+0.11 5.4_+1.1
Helianlhus
7 0.87±0.03 1.10±0.02 0.78±0.01 9.4±0.8 1.11_+0.11 0.43±0.05 0.24±0.01 2.5±0.3
21 0.92_+0.13 1.96-1-0.14 0.53±0.10 28.1_+2.0 3.66±0.50 1.69_+0.30 0.20_+0.04 5.8±0.6

Data are means_+S.D. (P ~< 0.05).

m e d i u m , the Pb content in the root was 11,247.6
mg kg ~ d r y wt and in the shoot 1400.3 nag kg
d r y wt). Comparisons of the total amounts of Pb
a c c u m u l a t e d in the shoots and roots tbr each of
the tbur species, much smaller differences. For
lead-tolerant tomato, a 9-tbld increase in Pb was
tound in the root relative to that in the shoot (19
#g in the root and 2.1/tg in the shoot); in the least
P b - t o l e r a n t species, sunflower, this difference was
only 1.3-Ibld (15.2/~g in the root a n d 11.7/~g in
the shoot). T h e a m o u n t of Pb retained in the root
in relation to total Pb taken up by the plant
was the lowest tbr sunflower (77.6 56.5%) and
highest tbr t o m a t o (81.7 90.1~}i,).
W h e n the a m o u n t of Pb in the m e d i u m was
increased tbur times (i.e. from 5 to 20 mg d m :~),
its concentration and total a m o u n t in the root of
sunflower did not change significantly, however,
root growth was completely inhibited (Fig. 1).
For P b - t o l e r a n t t o m a t o when the Pb con-
centration and its total a m o u n t in the root
increased, root growth continued (Fig. 1); a 4-
fold increase in Pb concentration in the m e d i u m
was a c c o m p a n i e d by a 2 4-fold increase in the
concentration aim total a m o u n t of Pb in shoots
of the e x p e r i m e n t a l plants ( T a b l e 2). Statistically
significant differences in the total amounts of Pb
a c c u m u l a t e d in shoots of these plants were tbund
at all applied Pb concentrations (Table 2). T h e
least tolerant sunflower and m u s t a r d (Fig. 1)
a c c u m u l a t e d more Pb in shoots than did flax and
t o m a t o (Table 2). However, the difl~rences in Pb
concentration in shoots of e x p e r i m e n t a l plants
were statistically significant only with the 5 mg
dm :~ Pb regimen.
Changes in the mineral content of plants during lead
uptake
Phosphorus. T h e phosphorus contents in shoots
m a i n t a i n e d at or slightly below control levels are
shown in Fig. 3. O n l y in s u n f o w e r shoot did P
significantly exceed the control level (when 20 mg
dm :3 Pb was used). In roots of all tbur plants, P
content was the same as in the control.
Calcium. T o m a t o , the most tolerant to Pb,
dif'ered significantly ti'om the other species tbr
tissue Ca content (Fig. 3). T h e Ca content in its
roots after 7 days of culture in the presence o f Pb
rose to levels exceeding controls by a b o u t 2-fold,
and in the shoots by a b o u t 50%. T h e shoot and
root Ca content in the other species r e m a i n e d
near or below control levels.
Magnesium. T h e M g content of the shoot did
not change very much with increase in Pb content
of the m e d i u m (Fig. 3). M g content remained
 MINERAL STATUS OF CROP PLANTS AND Pb TOLERANCE 579

a r o u n d control values in t o m a t o a n d mustard, to

T T 5 mo Pb' (Ira-a a rise in sunflower at 90 mg d m 3pb~+.

The response of experimental plants to mineral d@ciency

T h e degree of p l a n t tolerance to mineral defi-
cits was d e t e r m i n e d on the basis of d r y m a t t e r
xI~O u-O yield p r o d u c e d by the e x p e r i m e n t a l plants in
relation to controls. D r y m a t t e r yield was the least
inhibited in the most tolerant plants. T h e results
are presented in T a b l e 3.
In general, the concentrations of P, Ca and M g
in p l a n t tissues after 21 days of culture under
e x p e r i m e n t a l conditions of nutrient deficiency,
] ~'-"a Heti
Lycopersicon
on'~hus
&a~ varied in relation to control values in different
k species. I n general, the changes in elemental con-
stitution did not have a c o m m o n direction (either
--
~ / / ~ ~ "drn-3
increase or decrease) that was p r o p o r t i o n a l to the
degree of tolerance to the deficit of these elements
10C
(Fig. 4).
o

"6
,-A
b-x
a-O
a-x
/i--O
a-x /
°-'/
I ~-~
~-~
"4 I
Phosphorus deJMt. A decrease in tissue P con-
centration was p r o p o r t i o n a l to the degree of P-
a-o deficit tolerance. T h e most tolerant to P deficit,
flax (Table 3), h a d the smallest shoot and root
£ -
/ a-X ~ /
/I • d r o p in P level (in relation to control) (Fig. 4A).
,._. ./f'- g-; In terms of Ca and M g contents, the plants of the
four genera b e h a v e d differently. Flax, the most
tolerant to P stress (and second ill terms of Pb
tolerance) exhibited increased Ca and M g con-
centrations only in its shoot. In the most sensitive
species, t o m a t o (which is also the most tolerant
ID-x ~ 20rag Pb'dm-3 I
to Pb), the concentration of Ca and M g increased
significantly above control levels in shoots and
- I~-O roots. I n the r e m a i n i n g two species, M g and Ca
2o~ \ / r,_i~ ;'-6 =__-xr either did not change or decreased.
Calcium deficil. T o m a t o , the most Pb tolerant of
the species, was the most tolerant to Ca deficiency
days of cultivation (Table 3). Analysis of p l a n t mineral composition
(Fig. 4B) showed that P concentration did not
FIG. l. Changes in the tolerance index during constant
culture of plants in the presence of lead in the medium undergo significant changes in relation to control
in concentrations of 5, 10 and 20 nag dm :~. Bars rep- levels (with the exception of the increased P level
resent S.D. at P ~< 0.05 (n = 45). in flax shoots). However, a p r o n o u n c e d increase
in M g concentration was observed in all plants.
Magnesium deficit. M g deficiency caused a simi-
lar inhibition of growth a n d d r y m a t t e r yield in
all four species studied (Table 3). After 21 days
near control levels (flax, sunflower) or rose a b o v e o f c u h u r e the root and shoot P concentrations did
them (mustard, tomato). Changes in the root not differ fi'om control values (with the exception
m a g e n s i u m contents were also not uniform and of t o m a t o root) as was also the case with Ca in
ranged ti'om a large d r o p in flax, to oscillations mustard, sunflower a n d flax (Fig. 4C). In t o m a t o
580 D . M . ANTOSIEWICZ

shoot
Sinapis
Linum
Lycopersicon
x--x He[ianthus

o
E control
oo tevel +
D-A
5
E ._o
m0 - O a - a_ x

E)
u-O i

5C x-A .L /

A- x
&-O

Pb concentration in medium ( mg.dm -31

FIo. 2. Relative changes in the dry matter of the shoot and root (expressed
as oJ/o of control) after 7 days of cultivation in the presence of Pb at
concentrations of 5, 10 and 20 mg dm t. Bars represent S.D. at P ~< 0.05
(n = 3). Means differing in a statistically significant manner from each
other are marked on the graph in pairs using the same symbols for the
species as were used to draw the curves; means differing in a statistically
significant manner from the corresponding control are circled (Student's
t-test at P ~< 0.05). Genus (common name):Sinapis (mustard), Linum (flax),
Lycopersicon (tomato), Helianthus (sunflower).

and flax roots, however, the Ca content rose DISCUSSION

above control levels.
General nutrient stress. Flax, with its m o d e r a t e
constitutional tolerance to Pb, was the most tol-
e r a n t to general nutrient stress (Table 3). Dry
m a t t e r yield was the least inhibited in this species.
However, this highest tolerance was not accom-
panied by the highest concentrations of elements.
After 21 days of culture under the conditions of
this experiment, the plants showed significantly
higher P and Ca deficits in roots than in shoots
(Fig. 4D). In terms of M g contents, the t o m a t o
was r e m a r k a b l e for its large increase in M g con-
centration in its shoots and roots. Flax was also
noteworthy due to the decrease in its M g con-
centration in the shoot but increase in the root.
In the r e m a i n i n g species the M g content did not
change (mustard, sunflower).
The mineral status of plants and tolerance to Pb
T h e Wilkins test for tolerance (42i showed that
t o m a t o differed from the other three p l a n t species
in that it d e m o n s t r a t e d a statistically significant
high constitutional tolerance to Pb and was the
most tolerant of Ca deficiencies. These results sup-
port the hypothesis of a relationship between a
plant's Ca status, tolerance to Ca deficiency and
high constitutional tolerance to Pb.
Analysis of the P, Ca a n d M g contents in p l a n t
tissues conducted after 7 days of incubation in the
presence of PbC12 showed that t o m a t o had the
highest Ca content, increasing in roots (almost 2-
fold) a n d stems (about 4 0 - 5 0 % in relation to
controls; Fig. 3). In the species the least tolerant
to Pb, sunflower, the Ca content fell to below
control levels. KHAN a n d KHAN !26/obtained simi-
 M I N E R A L S T A T U S O F C R O P P L A N T S A N D Pb T O L E R A N C E 581

Table 2. Pb content of shoots and roots after 7 days of cultivation in the presence of Pb in the medium in concentrations of 5~
10 and20 mg dm - :~

Shoot Root

Pb
total amount
Pb PB Pb per 1 plant (as
concentration Pb total amount Pb total amount % of the total
in medium concentration per 1 plant concentration per 1 plant amount in the
(rag dm :~) Species* (mg kg i dry wt) (#g) (mg kg ~dry wt) (/~g) shoot + root)

Sinapis 466.8 __+84 b 4.1-t-1.5" 12,388.9___1198~b' 16.54-1.0 "u 80.1_+5.0

Linum 451.4 _-4-_90" 2.0_+0.4 "u 6107.2+_827 ~ 6.1 __.1.3"~ 75.3_+0.8"
Lycopersicon 307.1 ± 33"be 1.1±0.1 u 6500.7-1-23 b 4.9-t-0.1 ~ 81.7+0.9 ~
Helianthus 566.0 _+73~ 4.6-t-0.6 b 7173.3+1225 ~ 15.94-l.4 Cd 77.6-+2.4

10 Sinapis 907.2±257 7.5+1.8 ~u 23,506.0+2895 ~d 32.7__1.3 "b 81.3_+5.4"

Linum 729.9± 67 3.1 4-0.4" 14,011.9 ± 2204"b 14.0-+1.4 81.9+ 1.2b
Lycopersicon 580.9-+ 161 2.1 ___0.8u' 25,200.0-t-8025 b~ 19.0± 2.2~ 90.1 ±7.4 ~
Helianthus 811.9_+204 6.9__0.5 ~ 8415.3___ 2054~d 14.7+4.4 b 68.1_+8.5~b"

20 Sinapis 1459.5-+_ 518 11.0±3.2" 42,083.0+6660 ~b 39.4___5.2"h~ 78.24-2.8 a

Linum 1041.6+131 3.9-1-0.7" 19,953.5 ± 4093 b~ 19.9_2.4" 83.6-I-3.0 h
Lycopersicon 1380.9_+505 4.44-1.0 ab 23,841.3+3711 "" 19.1___3.8b 81.3±9.2"
Helianthus 1400.3-1-461 11.7+4.6 b 11,247.64-2854 ~ 15.2+4.4 ¢ 56.5-+ 13.6~u~

Values art: means of three experiments_+ S.D. at P ~< 0.05. In columns, means for every Pb concentration in medium fi)llowed
by the same letters are significantly different (using Student's t-test P ~< 0.05).
* Genus (common name):Sinapis (mustard), Linurn (flax), i:~coper~icon (tomato), Helianthus (suntlower).

lar results. T h e y d e s c r i b e d i n c r e a s e d C a levels in e l e v a t e d tissue C a levels. O t h e r e x a m p l e s of

the a b o v e - g r o u n d p l a n t parts o f the m o r e Pb-
t o l e r a n t t o m a t o in c o m p a r i s o n w i t h t h a t o f the
less t o l e r a n t p o t a t o . T h e results of o t h e r studies on
c u l t u r e d species (on so-called " s e n s i t i v e " species
h a v i n g o n l y a c e r t a i n d e g r e e o f c o n s t i t u t i o n a l tol-
e r a n c e to Pb) s h o w t h a t tissue C a levels fall, b u t
in c e r t a i n cases rise also, in response to a d m i n -
istration o f this m e t a l . (1°'12'21'4°'41)T h e s e studies did
not, h o w e v e r , assess the d e g r e e of t o l e r a n c e of
these plants to Pb. I n the p r e s e n t study it is s h o w n
t h a t in the p r e s e n c e of Pb, the C a level i n c r e a s e d
( t o m a t o - t h e most P b t o l e r a n t ) a n d also l o w e r e d
it or did n o t affect it at all in c o m p a r i s o n w i t h
controls (sunflower, m u s t a r d a n d f l a x - - l o w Pb
t o l e r a n c e ; Fig. 3). S i m i l a r l y , a p h y s i o l o g i c a l
mechanism permitting augmented Ca uptake
( w h e n its soil c o n c e n t r a t i o n was low) was f o u n d
by BABALONAS et al. (31 in plants g r o w i n g on soil
c o n t a m i n a t e d at n o r m a l l y toxic levels w i t h Pb,
Z n a n d C u (with i n d u c i b l e t o l e r a n c e to these
metals). T h e plants g r o w i n g on those sites h a d
a l t e r e d C a m e t a b o l i s m in a v a r i e t y e x h i b i t i n g
i n d u c i b l e t o l e r a n c e in c o n t r a s t to a n o n - t o l e r a n t
v a r i e t y w e r e d e s c r i b e d by F o Y e t a/. (17'1ai T o l e r a n t
s o y b e a n a n d s n a p b e a n varieties h a d the ability to
take up m o r e C a in the p r e s e n c e of A1, w h i c h led
to i n c r e a s e d C a c o n t e n t s in shoots a n d roots, T h e
n o n - t o l e r a n t varieties of these plants did not h a v e
such an ability a n d their r e d u c e d tissue C a con-
tent was associated w i t h their g r e a t e r sensitivity
to A1. D e t e r m i n a t i o n of the m e c h a n i s m ( s ) for
increased C a u p t a k e in the p r e s e n c e o f Pb by Pb-
t o l e r a n t t o m a t o (which the o t h e r plants w i t h a
low c o n s t i t u t i o n a l t o l e r a n c e to Pb did n o t have)
requires f u r t h e r study. T h e C a status o f the con-
trol t o m a t o plants in the present study is c h a r a c -
teristic for t o m a t o in general. It was s h o w n t h a t
after 7 days of g r o w t h in the absence o f P O 4 3 ions
(the Wilkins test c o n t r o l ) , the c o n t r o l t o m a t o
plants h a d the highest root C a c o n c e n t r a t i o n s
( T a b l e 1). I n turn, after 21 days o f g r o w t h u n d e r
the s a m e conditions, the r o o t a n d shoot C a levels
582 D.M. ANTOSIEWICZ

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 MINERAL STATUS OF CROP PLANTS AND Pb TOLERANCE 583

Table 3. A comparison of the degree of tolerance of the studied plants: ( i) to P, Ca, Mg and general nutrient
stress (estimated by dry matter yield of the plants, expressed as °/o of the control) and (ii) to the effect~ of l?b
(expressed as index of tolerance see Fig. 1)

General
P Ca Mg nutrient Action
Species* deficiency deficiency deficiency stress of Pb

Lycopersicon § ~ + + ~
S = 11+1 S= 88_7 S=61+7 S = 45_15
R = 17-t-2 R = 82+_8 R= 62_7 R = 116-t-19
Linum ~ + ++ t
S = 80___3 S = 60___8 S = 87-t-9 S = 70-t-4
R = 93___6 R = 71___4 R = 82+12 R = 98-1-7
Sinapis ++ § ~ + §
S = 51 -t-6 S = 29-t-6 S = 69-1- 14 S = 48-t-5
R = 73-t-8 R = 36-1-3 R = 70-t-12 R = 73-1-9
Helianthus .~ ~ + + §
S = 70+5 S=82+_1 S=81+6 S=57_11
R=80+5 R=53+5 R = 70+12 R = 56_7

Scale of tolerance: I", most tolerant; ,+, intermediate; §, most sensitive. Dry matter yield of the
plants: S, shoot; R, root. Data are means+ S.D. (P ~< 0.05).
* Genus (common name): Lycopersicon (tomato), Linum (flax), Sinapis (mustard), Helianthus
(sunflower).

in the tomato significantly exceeded control levels
(rig. 4A).
T h e results of the present study show that the
most P b - t o l e r a n t species, tomato (with high con-
stitutional Pb tolerance) is simultaneously the
most tolerant to Ca stress (Table 3). Development
of tolerance to m a c r o e l e m e n t deficits (which are
i n d u c e d by metal pollutants) occurs frequently in
plants from metal-tolerant lines with inducible
metal tolerance. For example, Al-tolerant lines of
wheat, barley a n d soya, in contrast to non-tol-
erant lines, possessed the ability to tolerate A1-
induced Ca deficiency, c19/ T h e i n d u c t i o n of tol-
erance to A1 is thus often associated with a certain
acquired change in mineral metabolism. It may
therefore be postulated that plants from a non-
polluted habitat, possessing an i n h e r e n t increased
tolerance to deficiencies of those macroelements
whose metabolism would be disturbed in the
experiment after administration of metal, could
also be characterized by an increased con-
stitutiona] tolerance to that metal. Thus, the tom-
ato in the present study d e m o n s t r a t e d high
constitutional tolerance to Pb, which was
accompanied not only by a characteristic elevated
tissue Ca content b u t by a tolerance to low Ca
stress. T h e role of Ca in alleviating p l a n t Pb tox-
icity has been postulated./3°/Therefore, it is poss-
ible that these characteristics not only help the
p l a n t to tolerate Pb, b u t also increase the plant's
constitutional tolerance to it. As can be seen from
the results of BEHLINO et al., (91 the characteristic

FIG. 3. The relative contents of P, Ca and Mg (expressed as % of control calculated from determined con-
centrations) in the shoot and root after 7 days of growth in the presence of Pb in the medium in concentrations
of 5, 10 and 20 mg dm -:~. Bars represent S.D. at P ~< 0.05 (n = 3). Means differing in a statistically significant
manner fi'om each other are marked on the graph in pairs using the same symbols for the species as were used
to draw the curves; means differing in a statistically significant manner from the corresponding control are
circled (Student's t-test at P ~ 0.05). Genus (common name): Sinapis (mustard), Linum (flax), lycopersicon
(tomato), Helianthus (sunfower).
584 D, M. ANTOSIEWICZ

D
150 A ii B

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shoot root shoot root shoot root shoot root

phosphorus / co[ciu m mognesium Knop's so(ution
deficiency j deficiency deficiency 50x dituted
 M I N E R A L STATUS OF CROP PLANTS AND Pb TOLERANCE 585

traits of a t o m a t o line unsusceptible to low Ca u p t a k e in the presence of A1 than that of the

stress (Ca-efficient line), in contrast to those of a
susceptible line (Ca-inefficient line), are m a i n l y a
high p r o p o r t i o n of soluble tissue Ca and an ability
to continue g r o w t h a n d d r y m a t t e r a c c u m u l a t i o n
u n d e r low concentrations of Ca. T h e distribution
a n d utilization of Ca in association with the effects
of Pb on the three species [tomato, sunflower (or
mustard) ], differing in their constitutional Pb tol-
erance a n d tolerance to low Ca stress in the pre-
sent study need to be elucidated.
T h e M g status of the t o m a t o was not as charac-
teristic as that of C a in association with Pb-tol-
erance. T h e M g c o n c e n t r a t i o n in control plants
after 7 days of growth (without P O ; 3 ions) was
very high in t o m a t o stems and in flax a n d t o m a t o
roots ( T a b l e 1). After 21 days of g r o w t h u n d e r P
deficit conditions, the M g level in comparison
with controls was highest in tomato, both in its
shoots a n d roots (Fig. 4A). However, in terms of
tolerance to M g deficit, the plants did not differ
from each other. It has been reported that Pb
has a negative effect on tissue M g c o n t e n t , ':13'21'41~
whereas in the present study, the effect of Pb
on tissue MK content was different tbr different
species, as tbr Ca content (Fig. 3). However, in
contrast to the results o b t a i n e d for Ca content,
increases in shoot M g levels a c c o m p a n y i n g Pb
a d m i n i s t r a t i o n were observed both in the most
P b - t o l e r a n t t o m a t o a n d the least P b - t o l e r a n t
mustard, as well as in the roots of the least Pb-
tolerant sunflower (at 20 mg d m -:~ Pb in the
m e d i u m ) . In the r e m a i n i n g cases, the level of M g
in the shoots and roots oscillated within control
limits or was lower. Therefore, on the basis of the
present results one c a n n o t conclude that the M g
status of" t o m a t o is associated with an increased
tolerance to Pb. I n contrast, the m e t a b o l i s m of
Ca a n d M g plays an i m p o r t a n t p a r t in tolerance
to A1; it has been shown that an A l - t o l e r a n t rye-
grass cultivar which evolved from a n o n - t o l e r a n t
" n o r m a l " p o p u l a t i o n , showed higher Ca and M g
n o r m a l population./31~,
T h e Wilkins root test (Pb tolerance test) is car-
ried out in the absence of P ions in the m e d i u m
because they form precipitates with Pb.(42~' A cer-
tain p a r a d o x arises from this because tomato, the
most P b - t o l e r a n t species according to this test is,
as the d a t a indicate, the most sensitive to P deficit
( T a b l e 3; the highest d r y m a t t e r loss in com-
parison with controls). W h y then was t o m a t o the
most sensitive to P stress alone while u n d e r con-
ditions of P a n d Pb stress, the most tolerant of the
four studied species? T h e observed reaction of
the plants was p r o b a b l y the result not of their
sensitivity to P deficit, b u t of their Pb tolerance.
T h e d u r a t i o n of the test was different in both
cases, 7 days for the Wilkins test and 21 days for
P stress. Seven days is too short a period for the
differences in tolerance of plants to P deficits to
become a p p a r e n t . However, in those cases in
which tolerance of various species of plants to Pb
is d e t e r m i n e d by cultivating them in the presence
of Pb for more than 7 days, it should be borne in
m i n d that the result m a y be influenced by the
various tolerances of the plants to P stress and
their variously affected mineral status.
T h e protective role of Ca against the toxic
effects of Pb has been suggested by RASHID and
PopovIc. (3°) I n their study it was shown that high
constitutional Pb tolerance was a c c o m p a n i e d
by such physiotypic features as tolerance to Ca
deficit and different Ca status, in comparison
with plants possessing low Pb tolerance. These
results do not mean, however, that ew:ry p l a n t
having these features will also have a high con-
stitutional Pb tolerance. It would therefore be
interesting to develop in t o m a t o (with high con-
stitutional Pb tolerance and high tolerance to
Ca deficit) a n d sunflower or m u s t a r d (with low
constitutional Pb tolerance and low tolerance to
Ca deficit) lines with high inducible tolerance
to Pb a n d c o m p a r e them with regard to their

FIG. 4. The relative P, Ca and Mg contents (expressed as °b of controls calculated ti~om determined con-
centrations) in the shoots and roots after 21 days of cultivation in (A) P deficit, (B) Ca deficit, (C) Mg deficit
and (D) medium diluted 50 x . Bars represent S.D. at P ~< 0.05 (n = 3). Treatments with common letters within
each group of shoots and roots are significantly different at P ~< 0.05 (without letters are not significantly
different); ( 0 ) significantly different from the corresponding control (using Student's t-test, P ~< 0.05). Genus
(common name): Sinapis (mustard), Linum (flax), l~copersicon (tomato), Helianthus (sunflower).
586 D.M. ANTOSIEWICZ
tolerance to low-Ca stress and associated Ca
status.
The content 0j'Pb in plants and tolerance lo Pb
In studies on tolerance, it is usually expected
that small differences in u p t a k e of metals by tol-
erant and n o n - t o l e r a n t genotypes m a y be a mani-
festation o f tolerance mechanisms. (7) In cases of
differences in the degree of constitutional tol-
erance a m o n g several species of plants, similar
relationships m a y also be expected. However, the
results of the present study have shown that there
is no p r o p o r t i o n a l i t y between the degree of con-
stitutional Pb tolerance a n d the Pb content of a
plant. T h e highest root Pb concentration was
tbund in mustard, the lowest in sunflower (Table
2) b u t both species were the least tolerant to Pb
of those studied (Fig. 1). I n turn, the total a m o u n t
of Pb taken up by the root and the entire plant
increased with increasing Pb concentration in the
m e d i u m , reaching its peak value in m u s t a r d at 20
mg Pb d m -:~ ( 3 9 . 4 / t g in the root; T a b l e 2). In
the r e m a i n i n g three p l a n t species, the initial high
a m o u n t of Pb taken up at a m e d i u m Pb con-
centration of 5 mg d m :~ was not affected at
m e d i u m Pb concentrations of 10 and 20 m g d m 3.
A t the same time, however, very large differences
in the reaction o f the plants to the different Pb in
root tissues were found. T h e T I s o f sunflower and
m u s t a r d were the lowest, although both represent
extremes in terms of a m o u n t a n d concentration
of Pb in their tissues (Fig. 1, T a b l e 2). F o r
example, for m u s t a r d a n d t o m a t o at 10 mg d m a
Pb in the m e d i u m , both h a d similar Pb con-
centrations a n d total amounts in roots whereas
the T I of m u s t a r d was very low (20%) and
that of t o m a t o very high (87°/o). A similar lack of
p r o p o r t i o n a l d e p e n d e n c e between the a m o u n t
of a c c u m u l a t e d Pb in the roots and the T I in
three species o f crop plants was also found by
BURZYNSVd.(t3) C o m p a r i s o n o f the u p t a k e of heavy
metals by tolerant a n d sensitive lines of the same
species has shown that the tolerant plants can
take up and a c c u m u l a t e in their roots more metal
than the n o n - t o l e r a n t lines. (]4'28'43) However,
opposite cases are also known in which, tbr ex-
ample, the level of the a c c u m u l a t e d metal is simi-
lar in both lines (14) or is higher in the roots of
sensitive plants relative to that in the tolerant
line.O 9)
T h e roots of the plants in the present study
retained a b o u t 80% of the total a m o u n t of Pb
taken up (the r e m a i n d e r was transported into the
shoots); this p r o p o r t i o n was m a i n t a i n e d for all
three Pb concentrations used in the m e d i u m
('Fable 2). Sunflower, the least P b - t o l e r a n t
species, was an exception in which the a m o u n t of
metal which entered the shoot reached a
m a x i m u m of 43.5% at the highest Pb con-
centration used. A barrier to Pb transport
through the p l a n t has been described. ':2~'29'44)An
a c c u m u l a t i o n in the roots of a p p r o x i m a t e l y 80o/o
of the Pb taken up is in a g r e e m e n t with the d a t a
of JONES ':25' tbr ryegrass. T h e exclusion of heavy
metals fi'om shoots is very c o m m o n in plants; such
plants are called "excluders". (4 T h e p a t t e r n of
Pb distribution in the plants used in the present
study indicates that they belong to excluders.
Preferential a c c u m u l a t i o n of Pb and other heavy
metals in roots has been observed in m a n y
plants..' 4,24,25,29,44=
T h e r e are, however, reports on the association
of increased tolerance to a metal with its
decreased translocation fi'om the root to the
shoots. '7'24) T h e destruction of the r o o t - s h o o t bar-
rier in excluders is considered to be indicative of
a threshold or greater effect of the metal which is
toxic to the plant. ~4' Therefore, the concentration
of the metal breaking this barrier should also be
used in d e t e r m i n i n g the level of tolerance. R o o t
growth was completely inhibited in mustard, flax
and sunflower by the Pb concentrations and
exposure durations used in the present experi-
ment, but in the least P b - t o l e r a n t species, sun-
flower, an increase in the a m o u n t of Pb in the
shoots, up to 43.5% of the total a m o u n t of Pb
taken up by the p l a n t (at a Pb concentration of
20 mg d m :~), was observed only in sunflower.
This result points to the destruction of the root
shoot b a r r i e r tbr Pb translocation. T h e dittkrence
in shoot Ph concentration between the least Pb-
tolerant sunflower a n d m u s t a r d is postulated to
be due to the fact that in spite of complete inhi-
bition of" root growth in mustard, the r o o t - s h o o t
b a r r i e r still had not been broken. In the most Pb-
tolerant tomato, however, a tendency to retain
the highest p r o p o r t i o n of Pb in the roots, relative
to that of the other species (81 82% at 5 and 20
mg Pb d m -3 and 90.1°/o at 10 mg Pb d m 3,
T a b l e 2), was a p p a r e n t . No significant differences
 M I N E R A L STATUS OF CROP PLANTS AND Pb TOLERANCE
a m o n g the studied plants were found at 10 and
20 mg d m 3 in the m e d i u m for shoot Pb con-
centrations.
T h e results of the present study show that the
root is the m a j o r organ in d e t e r m i n i n g survival
u n d e r conditions of substrate Pb pollution. T h e
low toxicity of a given dose of Pb, as has been
shown in numerous studies, 1529"44i is d e p e n d e n t
m a i n l y on the detoxification c a p a c i t y of the plant,
i.e. on its c a p a c i t y for sequestration of the metal
at the cellular level in places where it cannot
interfere with sensitive m e t a b o l i c activities. This
leads to the a c c u m u l a t i o n of metal in a detoxified
tbrm. Detoxification m a y result from cell wall
binding, active p u m p i n g of ions into vacuoles,
complexing by organic acids and by specifiic
m e t a l - b i n d i n g proteins. (5'29'39'44! O f course, each
p l a n t p r o b a b l y has its own limits tbr further
detoxification when the a m o u n t of Pb taken up
increases b e y o n d detoxification capacities. Each
p l a n t seems to possess a m e c h a n i s m controlling
the a m o u n t of Pb taken up (plants took up differ-
ent a m o u n t s of" Pb u n d e r the same conditions in
the present studies). I n this sense, constitutional
tolerance can d e p e n d on the a m o u n t of metal
taken up. Since one of the elements of the defense
of plants against Pb is its exclusion fi'om the
shoot, 4'7'~4' the b r e a k i n g of the r o o t - s h o o t b a r r i e r
could take place when the detoxification c a p a c i t y
of the root is surpassed.
After 7 days of growth in the presence of Pb,
t o m a t o h a d the highest root d r y m a t t e r yield (Fig.
2), exceeding that of control values. O n e of the
reasons tbr this m a y be increased t o m a t o root
thickness, but such m e a s u r e m e n t s were not made.
A n o t h e r possible e x p l a n a t i o n for the high yield in
d r y m a t t e r is that the presence o f h e a v y metals,
including Pb, in p l a n t tissues induces the synthesis
of l o w - m o l e c u l a r weight proteins which complex
these metals, thus limiting their toxic effects. '~7''*~
Such proteins are the functional c o u n t e r p a r t s of
metallothioneins identified in animals. Recently,
smaller peptides (called " p h y t o c h e l a t i n s " ) were
described in plants and p l a y an identical role in
detoxification. :~7'3~iIt c a n n o t be excluded that in
connection with the high Pb tolerance of tomato,
the synthesis o f c o m p o u n d s c o m p l e x i n g Pb is
more intense than in the r e m a i n i n g less Pb-
tolerant species; the presence of such complexing
substances would also c o n t r i b u t e to increasing the
587
root d r y m a t t e r content. A test of this hypothesis
requires further study. ROMAmUK a n d GABARAc~2
also believe that the increase in cytoplasmic d r y
m a t t e r of pea meristem cells after a d m i n i s t r a t i o n
of Cd, Pb and Cr was caused by the synthesis of
metallothioneins. A d m i n i s t r a t i o n of Zn, Cd and
H g also caused a slight increase in the protein
content leading to a rise in the d r y mass of
E u g l e n a cells.' i~
Acknowledgments I am grateful to the research group
of Prof. Andrze i Sapek for assistance in determinations
of elemental concentrations and amounts. "['he work
was supported by the State Committee for Scientific
Research, Poland, as grant KBN-4.0607.91.01.
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