Planta (2001) 212: 313322
Accumulation of heavy metals in epidermal glands
of the waterlily (Nymphaeaceae)
Noa Lavid1,*, Zahava Barkay2, Elisha Tel-Or1
1
Department of Agricultural Botany, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
2
Wolfson Applied Materials Research Center, Tel-Aviv University, Israel
Received: 29 April 2000 / Accepted: 8 June 2000
Abstract. This study investigates the anatomical aspects
of heavy-metal accumulation in the waterlily (Nymphaea
`Aurora', Nymphaeaceae). Epidermal glands were iden-
tied by light microscopy on the abaxial side of the leaf
laminae and on the epidermis of the rhizome; glandular
trichomes were observed in the petiole epidermis. Glands
were not observed in the roots. Accumulation of heavy
metals in these glands was monitored using a scanning
electron microscope equipped for energy-dispersive spec-
troscopy. Further experiments showed maximal cadmium
and calcium accumulation in the mature leaf lamina
in daylight, and this accumulation was inhibited by
the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea.
These results suggest that, in Nymphaea, heavy metals
are accumulated primarily in association with glands
found in plant organs that have direct contact with water
or mud. Deposition and storage of heavy metals by these
glands may represent a stage in the sequestration and
detoxication of the metals. Our results raise the possi-
bility of utilizing waterlilies for the removal of heavy
metals from polluted environments.
Key words: Cadmium accumulation Glandular
trichome Heavy metal Nymphaea (Cd accumulation)
Phytoremediation
Introduction
Soil and water pollution by toxic heavy metals has
become a major environmental concern. Thus the use
*Present address: Faculty of Agricultural,
Food and Environmental Quality Sciences, The Hebrew University
of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
Abbreviations: BSE back-scattered electron, DCMU 3-(3,4-
dichlorophenyl)-1,1-dimethylurea, EDS energy dispersive spec-
troscopy, ICP inductively coupled plasma, SE secondary
electron, SEM scanning electron microscopy
Correspondence to: N. Lavid;
E-mail: telor@agri.huji.ac.il; Fax: +972-8-9467763
of metal-accumulating plants for environmental phy-
toremediation has become established as a new
technology and scientic discipline (Salt et al. 1995a).
Because most industrial and municipal waste is
disposed of in water bodies and since certain aquatic
plants ourish in environments polluted by heavy
metals (Reimer and Duthie 1993), these plants have
been studied for their potential use in wastewater
treatment.
Under native conditions, the rhizome and roots of the
waterlily (Nymphaea) are planted in the mud and its
laminae, carried on long petioles, oat on the water's
surface. These plants are not only tolerant of high levels
of toxic heavy metals but they can also accumulate them
in large amounts (Klavins et al. 1992; Pip and Stepaniuk
1992; Reimer and Duthie 1993; Twining 1993; Quirez
and Miranda 1994).
Studying Nymphaea violacea, Twining (1993)
observed signicant foliar accumulation of radium and
calcium (Ca) with only slight translocation of the
accumulated metals; in addition, the degree of metal
uptake tended to increase with tissue age. Early eld
observations and glasshouse experiments performed in
our laboratory revealed that the laminae, petioles,
rhizomes and roots of waterlilies accumulate cadmium
(Cd) independently and directly from the liquid phase
(T. Schor, personal communication). No evidence was
found for Cd translocation from roots to leaves.
Localization and accumulation of heavy metals in
Nymphaea plant cells and organs have not been inves-
tigated extensively and specialized organs, tissues or cells
responsible for metal accumulation have not been
identied.
This study was aimed at investigating heavy-metal
uptake, accumulation and localization in Nymphaea.
Employing several heavy metals, but specically Cd, a
model was designed to elucidate this process. A study
focusing mainly on the structure and function of the
epidermal glands of the waterlily was designed to reveal
the mechanism by which these aquatic plants are able to
accumulate heavy metals and maintain a high tolerance
to their toxicity.
314
Materials and methods
Plant material and growth conditions
Waterlily plants are vegetatively reproduced and can grow and
survive for several weeks on their rhizome content. The commercial
species of Nymphaea `Aurora' (dened by The International
Water Lily Society, 1993) was used in all the experiments. Rhizome
explants of waterlily (obtained from Kibbutz Hazorea, Israel),
carrying regeneration buds, were grown during the spring and
summer in an unheated glasshouse under natural long-day condi-
tions (1014 h light). Each explant was planted in a pot (5 cm
diameter, 7 cm height). For experiments on the eect of Cd on
mineral content, a heavy clay soil (Kibbutz Hazorea, Israel) was
used. For experiments on Cd uptake by all plant tissues, a gravel
medium was used. Each pot was placed in a black, cone-shaped
(15 cm diameter at the bottom, 30 cm diameter at top, 26 cm
height) 10-l pot, containing 100 mg/l Ca in tap water. Three well-
developed mature plants were exposed to 50 mg/l Cd (as
Cd(NO3)2) or 50 mg/l Cd + 500 mg/l Ca (as CaCl2) added to
the water medium. Control plants were grown without supplemen-
tary Cd or Ca. Calcium chloride was used in the growth medium
for this experiment since early eld observations revealed that
waterlilies accumulate more heavy metals in saline water containing
high levels of CaCl2. Cadmium at a concentration of 50 mg/l was
used, as this is sucient for scanning electron microscopyenergy
dispersive spectroscopy (SEM-EDS) and for tracking Cd accumu-
lation. Other treatments included the addition of 500 mg/l Ca,
50 mg/l Fe (as FeCl3), 50 mg/l Pb (as Pb(NO3)2) or 2 mg/l Hg (as
HgCl2) to the water medium of plant containers. These concentra-
tions were chosen to represent those found in sludges in Israel and
to allow normal growth of the plant. Samples for microscopic
analysis were taken at predetermined times after metal application.
Only mature plant leaves exposed to 2 mg/l Hg exhibited some
damage, while young new leaves from this treatment did not, nor
did any plant tissues from any of the other heavy-metal treatments
during the subsequent 3 weeks. Dark treatments were conducted in
containers covered with aluminum foil. Three well-developed,
mature plants were exposed to 50 mg/l Cd (as Cd(NO3)2) or 50 mg/
l Cd + 10)5 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
added to the growth medium and were compared to control plants
grown in growth medium in the absence of Cd and DCMU. No
toxic eects were observed.
Plant sampling: quantication
The whole plant was dipped in Decon 90 (0.1%, v/v; Decon
Laboratories, Hove, East Sussex, England), washed with deionized
water, dipped in HCl (0.1%), and washed three times with
deionized water. Each plant was then separated into roots, rhizome
epidermis, rhizome medulla, petioles and leaf laminae. Very young
and senescent tissues were discarded. ``Mature leaf'' was dened by
a fully expanded lamina for a minimum of 2 weeks; ``young leaf''
was dened by a partially expanded, unrolled lamina.
Analytic microscopy
Plant tissues were harvested from three similar treatments, washed as
above followed by three washes with double-distilled water (ddw).
Tissues were sectioned with a sharp razor blade and washed once
more with ddw. Leaf sections or epidermal peels were immediately
attached to scanning electron microscope slides with double-sided
sticky tape, frozen ()70 C) and lyophilized for up to 3 h, in order to
preserve the natural tissue structures. Leaf samples were observed by
SEM and samples were chosen for photographic documentation.
The leaf residues underwent heavy-metal assay by inductively
coupled plasma (ICP) spectroscopy (Spectro, Kleve, Germany).
N. Lavid et al.: Glands of Nymphaea accumulate metals
An Olympus model BH-2 microscope was used to examine
plant sections by light microscopy. Carbon-coated samples were
examined in a JSM-6300 scanning electron microscope equipped
for Link energy dispersive spectroscopy (EDS). The resolution of
the EDS, as dened at Mn Ka FWHM (full-width half-maximum),
was 138 eV. Mass sensitivity of the spectroscope was 2,000 mg/l.
The EDS software used was Link ISIS (Oxford). The scanning
electron microscope was operated simultaneously in three modes:
(a) secondary electron (SE) mode, used to obtain the sample's
surface structure; (b) back-scattered electron (BSE) mode, used to
distinguish between the heavy metal accumulated and the sample
background; (c) EDS mode. The EDS system measures character-
istic X-rays emitted from the elements in the sample, in order to
determine its chemical composition, and the spectra show X-ray
counts versus the element's characteristic X-ray energy. Spectra
were compared under the same working conditions of electron
beam voltage (20 kV), magnication and counts per second (CPS).
X-ray mapping of certain elements was performed in addition to
the EDS analysis. Penetration depth is a few microns for light
elements down to submicrons for heavy elements at an electron
beam of 20 kV. The exact value depends on the specic concen-
tration of elements and can be estimated to be in the order of 1 lm.
The heavy metals investigated were Cd, Ca, Mn, Fe, Pb, and Hg.
The background elements were C, O, Cl, S, Si, Na, K, and P. The
main X-ray lines of Cd (L-line at 3.134, 3.317, 3.528 keV), Mn
(K-line at 5.9 keV), Fe (K-line at 6.2, 6.5 keV) and Pb (M-line at
2.4 keV) are well separated from the background elements, C
(K-line at 0.277 keV), O (K-line at 0.525 keV), Cl (K-line at
2.621 keV), Na (K-line at 1.041 keV), Si (K-line at 1.739 keV), K
(K-line at 3.312 keV), P (K-line at 2.013 keV). The main Ca K-line
overlaps the small Cd line at 3.7 keV. In order to distinguish
between the two, we correlated the 3.1-eV region with Cd, and the
3.7-eV region with Ca. Similar overlap exists between the S K-line
and the Hg M-line at about 2.3 keV, which may correspond to
HgS crystals as well as to Hg alone, as found by ICP spectroscopic
metal analysis of these leaves. Such HgS crystals were observed by
Barnett et al. (1997). Although data from X-ray analyses cannot be
considered a reliable indicator of global composition, it is
presented here as qualitative information on the local elemental
composition.
Metal analysis
Metal content of the plant samples was determined in oven-dried
material (80 C, 48 h). The samples were hand-milled, mixed to
homogeneity and weighed aliquots (300 mg) were digested in
10 ml concentrated HNO3 by heating (300 C, 10 min). After
cooling, the digest was diluted to 100 ml with ddw, ltered through
Whatman No. 1 paper and analyzed for metal content by ICP
spectroscopy.
Statistics
Each treatment was conducted in three replicates and each
experiment was conducted at least three times. Experiments were
performed at dierent times in a glasshouse under natural
conditions and therefore dierences among absolute values were
quite large. Nevertheless, the trends were similar in all experiments,
and representative experiments are therefore presented to express
both trends and variation in absolute values. Each experiment was
analyzed by Analysis of Variance (ANOVA; three treatments) or
by t-test (two treatments). Because of heterogeneity of variance,
some data were transformed logarithmically before the statistical
analysis. In the case of three treatments, pairwise comparisons were
performed by Tukey test (P 0.05). Two-way ANOVA was used to
analyze dierent aspects of the data on Cd treatment, leaf age and
exposure time (Table 2).
N. Lavid et al.: Glands of Nymphaea accumulate metals 315

Table 1. The eect of Ca on Cd accumulation in mature Nym- tissues is expressed in mg/g DW, mean SE, n = 3. Treatment
phaea plants. Plants were grown on gravel and exposed to means with dierent letters dier signicantly by the Tukey test
Cd(NO3)2 and CaCl2 for 48 h as indicated. The Cd content in the (P = 0.05)

Treatment Root Rhizome Leaf

Epidermis Medulla Petiole Lamina

Control, water 0.04 0.01 b 0.02 0.01 b 0.01 0.00 a 0.01 0.00 c 0.03 0.01 c
Cd (50 mg/l) 3.18 0.19 a 0.36 0.07 a 0.01 0.00 a 1.46 0.14 b 2.49 0.09 b
Cd (50 mg/l) + Ca (500 mg/l) 4.71 0.27 a 0.76 0.04 a 0.01 0.00 a 4.40 0.22 a 8.62 0.61 a

Table 2. Eect of leaf age and time of exposure to 50 mg/l Cd on between columns in the same row for a given metal. Dierent ca-
metal accumulation in the leaf laminae of waterlily grown on soil. pital letters denote signicant dierences (P < 0.05) between rows
The metal content is expressed in mg/g DW, mean SE, n = 3. of the same column for a given metal
Dierent small letters denote signicant dierences (P < 0.05)

Metals Exposure Young Young Mature Mature

analyzed time to lamina, lamina, lamina, lamina,
in lamina Cd (h) control 50 mg/l Cd control 50 mg/l Cd

Cd 0 0.03 0.01 aA 0.03 0.02 aC 0.02 0.01 aA 0.04 0.01 aC

7 0.04 0.01 cA 0.94 0.09 bB 0.04 0.01 cA 2.68 0.24 aB
48 0.04 0.02 cA 3.86 0.43 bA 0.03 0.02 cA 8.10 0.22 aA
Ca 0 4.00 0.39 cA 4.58 0.46 cB 11.46 0.97 bA 14.32 1.81 aB
7 4.10 0.19 dA 5.39 0.10 cB 11.82 0.81 bA 15.12 0.60 aB
48 4.50 0.38 dA 10.13 0.41 cA 12.02 1.04 bA 21.04 1.36 aA
Mn 0 0.10 0.01 bA 0.10 0.01 bA 2.37 0.22 aA 2.16 0.19 aA
7 0.10 0.01 cA 0.06 0.01 dB 2.38 0.18 aA 0.30 0.02 bB
48 0.11 0.02 cA 0.07 0.01 dB 2.40 0.19 aA 0.19 0.02 bB

Results Table 3. The eect of light and 50 mg/l Cd on metal accumulation

Nymphaea plants grown in water in the presence of
50 m/l Cd accumulated substantial quantities of Cd in
their roots, petioles and leaf laminae, but considerably
less in the rhizome (Table 1). The addition of 500 mg/l
Ca+2 to the medium increased Cd accumulation in
laminae and petioles about 3-fold (P < 0.05).
Table 2 displays the eects of leaf age and time of
exposure to 50 mg/l Cd on metal content in the laminae
of plants grown in soil. The metal content of control
laminae after 0, 7, and 48 h did not change. Young
treated laminae accumulated signicantly less Cd
(P < 0.0001) than mature treated laminae after 7 and
48 h of exposure to 50 mg/l Cd. Temporal uptake
studies revealed Cd accumulation to be a rapid process.
Considerable quantities of Cd were taken up during the
rst 7 h of the experiment and continued to increase
after 48 h of exposure (P < 0.0001). Manganese
content, which originated from the clay-type soil, was
higher in mature and young control laminae and
diminished signicantly (P < 0.0001) during Cd up-
take. The Mn content in control and Cd treatments
after both 7 and 48 h was signicantly higher
(P < 0.0001) in mature laminae. The concentration of
Mn in Cd-treated laminae did not change signicantly
between 7 and 48 h.
Calcium was accumulated together with Cd in young
and mature laminae and was higher in Cd-treated plants
than in control plants (P < 0.001). Mature leaves
showed a tendency to accumulate more Ca than young
in mature leaf laminae of Nymphaea plants. Plants were grown on
soil and exposed to Cd and darkness for 48 h as indicated. Light
conditions consisted of natural lighting of 1014 h duration. The
metal content is expressed in mg/g DW, mean SE, n = 3.
Treatment means with dierent letters dier signicantly by the
t-test (P = 0.05)
Light Metals analyzed in laminae
regime
Cd Ca Mn
Dark 1.73 0.33 b 15.62 2.25 a 1.14 0.06 a
Light 10.20 0.36 a 24.00 2.69 a 0.29 0.07 b
leaves (P < 0.0001) but 48 h after initiation of Cd
treatment, the dierence in Ca content between 7- and
48-h treatments in mature laminae was less than in
young laminae (P < 0.01).
Accumulation of Cd by the Nymphaea leaf lamina
was enhanced nearly 6-fold by light (Table 3,
P < 0.0001). Similarly, the Mn content in the dark
and light treatments diered signicantly (P < 0.0008),
suggesting that Mn is depleted due to Cd accumulation
in the light. The Ca content did not dier between
treatments. Calcium appeared to be accumulated with
age, and more rapidly in the presence of Cd.
3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibi-
tor of photosynthetic electron transport, inhibited Cd
accumulation 3-fold in the photosynthetic tissues of the
leaf, but not in the non-photosynthetic tissues of the
rhizome (Table 4).
316 N. Lavid et al.: Glands of Nymphaea accumulate metals

Table 4. Eect of DCMU on Cd accumulation in mature Nym- Ca accumulation, which also occurs in the absence of
phaea plants. Plants were grown on gravel under light conditions Cd; and Cd accumulation is accompanied by Mn
and treated with DCMU and Cd for 48 h as indicated. The Cd depletion and is more pronounced in mature leaves.
content is expressed in mg/g DW, mean SE, n = 3. Treatment
means with dierent letters dier signicantly by the t-test Analysis by SEM-EDS demonstrated selective accu-
(P = 0.05) mulation of Cd in specic loci in the abaxial epidermis of
the mature laminae (Fig. 1A). A representative X-ray
Treatment Lamina Petiole Rhizome emission spectrum (Fig. 1B) of a single locus shows the
epidermis high content of Cd and Ca, while neighboring cells
50 mg/l Cd 4.37 0.14 a 4.59 0.23 a 0.37 0.03 a contained only minor amounts. Untreated plants
50 mg/l 1.59 0.04 b 1.14 0.03 b 0.38 0.10 a (Fig. 1C,D) showed no such accumulation. This obser-
Cd + 10)5 M vation was veried quantitatively by ICP spectroscopy
DCMU (4.4 mg/g Cd, 20.2 mg/g Ca compared to control values
of 0.0 mg/g Cd, 9.0 mg/g Ca).
The experimental results led to the following obser- The abaxial epidermis of the lamina and the epider-
vations: Cd accumulation is dependent on leaf age and mis of the petiole and rhizome contain glands or
time of exposure to this metal; light conditions positively trichome glands in organized structures (Fig. 2AC). A
aect Cd uptake; Cd accumulation is accompanied by comparative analysis of Cd-grown plants by SEM-EDS

Fig. 1AD. Images of epidermal glands on the laminae of mature treatment. B Spectrum of a single gland. C General view of an
Nymphaea leaves, as yielded by SEM (A, C) and EDS spectra (B, D). untreated lamina. D Spectrum of a single gland in C. CPS, counts per
A General view of the abaxial epidermis after 24 h of 50 mg/l Cd second
N. Lavid et al.: Glands of Nymphaea accumulate metals
Fig. 2AC. Light microscopy of epidermal glands of Nymphaea. A Abaxial epidermis of a lamina. B Petiole epidermis. C Rhizome epidermis
demonstrated that all of these glands accumulate Cd,
particularly those in the abaxial epidermis of the lamina
(Fig. 1A) and of the petiole. Figure 3 shows Cd accu-
mulation in the petiole (4.1 mg/g as determined by ICP).
Figure 3A is an overall picture of the petiole and 3B is
an enlargement, displaying a single trichome. Trichomes
accumulated Cd in their basal glands, not in the hair
itself (spectral data are not shown). Epidermal glands on
the rhizome contained the lowest levels of Cd (not
shown).
Cadmium accumulation and mobilization inside the
leaf lamina is shown in Fig. 4A. The Cd was taken up
through the lower epidermis and after 24 h it had
reached the upper epidermis. An ICP analysis demon-
strated a concentration of 6.2 mg/g Cd (spectral data
not shown). Most of the Cd was immobilized in the
lower epidermis. Figure 4B shows the overall metal
distribution in the peripheral epidermis and parenchyma
of the petiole, but not in the central part of the
aerenchyma. Cadmium concentrations were determined
by ICP and found to be 5.6 mg/g (mapping data not
shown).
Cadmium accumulation accompanied by Ca accu-
mulation was observed at the whole-organ level. Addi-
tion of Ca+2 to the growth medium increased Cd
accumulation in the laminae and petioles (Table 1).
Figure 5AC shows that these two metals are co-
localized in abaxial epidermal glands (8.4 mg/g Cd,
24.8 mg/g Ca as determined by ICP). The simultaneous
accumulation of Cd and Ca was characterized by a
typical crystalline structure in the leaf epidermal gland
cells (Figs. 1A, 3) and within the leaf (Fig. 4). The
crystal (shown at high magnication in Fig. 6AC)
formed a square structure 2 lm in length and con-
tained both Cd and Ca (Fig. 6D).
317
Additional preliminary studies were conducted in
order to determine whether this pattern of Cd accumu-
lation by the leaf epidermal glands is common to other
heavy metals such as Fe, Pb and Hg. Accumulation of
Fe in the laminar epidermal gland cells was accompa-
nied by Mn (Fig. 7AC). Spectral analysis (Fig. 7D)
demonstrated low amounts of Ca, which may have been
depleted during Fe uptake (4.7 mg/g Fe, 1.9 mg/g Mn,
6.8 mg/g Ca, compared to control values of 0.2 mg/g Fe,
0.9 mg/g Mn, 12.2 mg/g Ca, as determined by ICP).
Accumulation of Pb in a single gland is shown in
Fig. 8A. Spectral analysis (Fig. 8B) showed that Ca and
Mn accompanied Pb (0.9 mg/g Pb, 12.8 mg/g Ca,
0.4 mg/g Mn, compared to control values of 0.0 mg/g
Pb, 11.2 mg/g Ca, 0.2 mg/g Mn, as determined by ICP).
Accumulation of Hg in a single gland is shown in
Fig. 9A. The spectrum of this gland (Fig. 9B) shows a
depletion of Mn and Ca during Hg uptake and
accumulation (0.9 mg/g Hg, 0.1 mg/g Mn, 10.1 mg/g
Ca, compared to control values of 0.0 mg/g Hg, 0.2 mg/
g Mn, 13.1 mg/g Ca, as determined by ICP).
Discussion
In this study, we describe the anatomical aspects of
heavy-metal accumulation in the waterlily and the roles
of light, tissue age and Ca in this accumulation. We
demonstrate that epidermal glands on the submerged
surfaces of waterlily leaf laminae, petioles and the
rhizome accumulate heavy metals.
Laminar glands have been previously reported in
waterlilies (Conard 1905; Luttge and Krapf 1969) and
similar structures, designated hairy or trichome glands,
have been identied in the petioles (Conard 1905). Our
318
Fig. 3A,B. Epidermal glands on a mature petiole of Nymphaea after
48 h of 50 mg/l Cd treatment, as seen by SEM. A A BSE image of
petiolar epidermal glands. B An SE image of a single petiolar
epidermal gland
observations demonstrate that such glands are also
present in the rhizome.
Luttge (1964) and Luttge and Krapf (1969) described
epidermal glands as consisting of the following four
cells: an upper cap that becomes extremely permeable to
water and salts and degenerates rapidly during leaf
development; two glandular cells, rich in mitochondria
and plasmodesmata, which seem to play an active role in
the transport of water and minerals; and a ``socket cell''
which resembles the mesophyll cells. The role of these
glands in metal accumulation has not yet been demon-
strated.
The metal accumulation observed here appears to
occur on and around degenerated cap cells. This
description concurs with the observation that not all
the glands in a single leaf appear to accumulate metals
(Fig. 1A). Mature leaves possess larger numbers of
degenerated cap cells (Kristen 1969) and this would
explain why the process of metal accumulation is
accelerated as leaves age. The special staining and
N. Lavid et al.: Glands of Nymphaea accumulate metals
Fig. 4A,B. Cross-sections of a mature leaf from a Nymphaea plant
treated for 24 h with 50 mg/l Cd, as seen by BSE imaging. A Leaf
lamina. B Petiole
plasmolysis methods used to determine the viability of
these gland cells before or during metal uptake failed to
yield a clear answer. To determine the status of the cells
during various stages of the process, further studies of
cross-sections under a transmission electron microscope
equipped with EDS (TEM-EDS) are required.
The function of these glands remains unclear. Kristen
(1969) concluded that their primary function is not the
uptake of water and minerals, but the secretion of
mucilage, which is a factor involved in the unfolding and
expansion of the young leaf. Only after mucilage
secretion ends, during leaf maturation, does the gland
become capable of water and mineral uptake. Thus,
mature leaves are expected to accumulate more metals
than young ones, as found here (Table 2). Since a similar
gland structure was also observed on the epidermis of
the petiole and rhizome, it seems unlikely that mucilage
secretion is the only role played by these glands. Similar
to other aquatic plants, waterlilies have reduced xylem
vessels (Fahn 1989); thus these epidermal glands might
function in the uptake of mineral nutrients and water.
N. Lavid et al.: Glands of Nymphaea accumulate metals
Fig. 5AC. The abaxial epidermis of a Nymphaea leaf lamina after 48
h of 50 mg/l Cd treatment. A An SE image. B Cadmium X-ray
mapping. C Calcium X-ray mapping
Fig. 6AD. A single crystal (2 lm 2 lm) from a Nymphaea leaf
lamina epidermal gland, as shown by SE imaging (A), Cd X-ray
319
mapping (B), Ca X-ray mapping (C), and EDS spectroscopy (D)
Fig. 7AD. Accumulation of Fe in the epidermal glands of a
Nymphaea leaf lamina. A A BSE image. B Manganese X-ray
mapping. C Iron X-ray mapping. D An EDS spectrum of one gland
320 N. Lavid et al.: Glands of Nymphaea accumulate metals

Fig. 8A,B. Accumulation of Pb in epidermal gland of a Nymphaea leaf lamina as shown by BSE imaging (A) and EDS spectroscopy (B)

Fig. 9A,B. Accumulation of Hg in epidermal gland of a Nymphaea leaf lamina as shown by BSE imaging (A) and EDS spectroscopy (B)

Typical co-crystallization of Cd and Ca (apparently
due to their similar atomic radius) accumulating in
epidermal glands results in Mn depletion. Salt et al.
(1995b) described the depletion of Mn as a result of
Cd uptake in the Indian mustard plant; however, this
relationship was not studied further. Accumulation of
Mn is a typical phenomenon that we have also
observed in late-season mature leaf laminae main-
tained under eld conditions. Specic co-precipitation
of elements, sometimes causing depletion of other
elements, was also shown for Fe, Pb and Hg
accumulating in laminar epidermal glands. However,
the nature of their co-precipitated crystals remains to
be claried.
The spectral methods that were available to us do not
allow a determination of whether and what organic
materials are involved in what appears to be biological
crystallization of heavy metals in plants. Mazen and El
Maghraby (1997) described Cd and Pb accumulation in
the form of Ca-oxalate crystals found on the aquatic
plant Eichhornia crassipes. We did not nd any Cd on
the Ca-oxalate crystals which are known to exist in
sclereides and which are abundant in Nymphaea. The
Cd-Ca-oxalate crystals in E. crassipes were completely
dierent in size and shape relative to the Nymphaea
Cd-Ca crystals. Crystals of Cd (Holmes et al. 1997) and
Ag (Klaus et al. 1999) have been shown to form in the
periplasm of bacteria via biological activity, and these
forms dier from non-biologically formed crystals.
Kroger et al. (1999) showed in diatoms that the typical
silicon structures are formed by involvement of specic
poly-cationic peptides. The phenomenon of special
N. Lavid et al.: Glands of Nymphaea accumulate metals
biological creation of metal crystals is a process that
should be examined more closely.
Translocation of Cd was not observed in the plant.
Cadmium is apparently immobilized in the epidermal
glands in the early stages of heavy-metal accumulation,
and subsequently in other epidermal cells. This immo-
bilization would explain the waterlily's high tolerance to
heavy metals.
Accumulation of Cd in the waterlily was enhanced by
light (Table 3). It appears that photosynthetic energy or
products may be involved in heavy-metal uptake by the
waterlily epidermal glands, as metal uptake was lower in
the dark, and green tissues in the leaves (laminae and
petioles) accumulated a higher level of metals than the
non-green rhizome tissues. In addition, inhibition of Cd
accumulation in the light was observed in the presence of
DCMU (Table 4). This is in agreement with Luttge
(1964) and Luttge et al. (1971), who demonstrated that
waterlily glands accumulate more radioactive sulfur (as
SO4 2 ) than adjacent epidermal cells and that this
accumulation is dependent on metabolic energy.
Secretory glandular trichomes occur in many families
throughout the plant kingdom. The trichomes show a
great variation in shape, structure and biochemical
capacity (Fahn 1979). Glandular trichomes usually
secrete directly into the external environment. The
waterlily epidermal glands dier in that they accumulate
metal ions as they mature. Cadmium, taken up by the
roots, accumulates preferentially in the trichomes on the
leaf surface of Indian mustard (Salt et al. 1995b),
suggesting that Cd storage in the trichome is a detoxi-
cation mechanism. Trichomes in other plants have been
reported to accumulate toxic ions, including Mn (Blamey
et al. 1986) and Pb (Martell 1974; Tung and Temple
1996), or to regulate Ca storage and its release (De Silva
et al. 1996). Expression of a type-2 metallothionein, a
metal-binding protein, in trichomes of bean plants has
been suggested to be a mechanism for heavy-metal
tolerance (Foley and Singh 1994). Secretory trichomes in
the aquatic plant Myriophyllum spicatum L. have been
reported to release allelopathic hydrolyzable polyphenols
(Godmaire and Nalewajko 1990; Gross et al. 1996). Such
polyphenols may chelate and detoxify heavy metals
within the glands (Lavid et al. 2000).
This study describes the incorporation and accumu-
lation of heavy metals in Nymphaea. These metals were
shown to be localized and immobilized in the epidermal
glands during the early stages of toxic heavy-metal
accumulation. Such structural studies contribute to our
understanding of the mechanisms by which waterlilies
accumulate heavy metals. The role of tissue age and
calcium in this mechanism and the basis for high
tolerance to toxic heavy metals were also examined.
Further experiments performed with industrial sludge
(200 mg/l Cd) and municipal sludge (4080 mg/l Cd)
will help evaluate the potential of waterlilies for the
commercial control of heavy-metal pollution.
The authors are grateful to Professors A. Fahn (Department of
Botany, The Hebrew University of Jerusalem) and M. Fridlander
(Israel Oceanographic and Limnology Research Ltd., Haifa) for
321
their most valuable suggestions, and to Professor D. Koller
(Department of Botany, The Hebrew University of Jerusalem)
and Dr. E. Lewinsohn (ARO, Newe Ya'ar Research Center, Ramat
Yishay, Israel) for their editorial contribution.
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