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Annals of Botany 86: 73±78, 2000
doi:10.1006/anbo.2000.1161, available online at http://www.idealibrary.com on
Relative Abundance of Nickel in the Leaf Epidermis of Eight Hyperaccumulators: Evidence
that the Metal is Excluded from both Guard Cells and Trichomes
G . K . P S A R A S *{, T H . CO N S TA N T I NI DIS{, B. COT S O PO U LO S{ and Y. M A N E TA S {
{Section of Plant Biology, Department of Biology, School of Sciences, University of Patras, Patras 265 00, Greece
and {Laboratory of Electron Microscopy and Microanalysis, School of Sciences, University of Patras,
Patras 265 00, Greece
Received: 15 November 1999 Returned for revision: 2 February 2000
Accepted: 16 March 2000
Scanning electron microscopy combined with X-ray microanalysis was used to localize the sites of nickel accumulation on the leaf epidermis of eight nickel accumulators grown on ultrama®c soils in Greece. In all species, nickel was
excluded from guard cells, and in species possessing hairy leaves (seven out of eight) nickel was excluded from the
hairs. In some species the metal was present in subsidiary cells, yet at low levels, while the sites of higher accumulation
were epidermal cells away from stomata. Results indicate that nickel is not compatible with the functions and
# 2000 Annals of Botany Company
development of certain epidermal cell types.
Key words: Brassicaceae, epidermis, metal hyperaccumulators, microanalysis, serpentine plants.
I N T RO D U C T I O N
Although some heavy metals are essential trace elements for
plant life, at relatively high concentrations they are toxic
since they interfere with enzyme function (De Vos et al.,
1989; Krupa et al., 1993). Accordingly, ultrama®c soils rich
in such metals are considered to be hostile, and they often
support a specialized ¯ora (Brooks, 1998a) of metal
resistant plant species (metallophytes). Although, in many
cases, resistance is due to exclusion of the metal from the
protoplast (Ernst, 1976), some plants actively take up
metals leading to accumulation at extremely high levels, far
exceeding those in the soil (Brooks, 1998b).
Hyperaccumulation raises interesting biological questions such as the mechanisms by which toxicity is avoided
and the possible adaptive signi®cance of such high levels of
heavy metals. With regards to nickel hyperaccumulators, it
has been reported that the absorbed metal is rendered
inactive by complexing with histidine (KraÈmer et al., 1996),
other amino acids and carboxylic acids including malate
(Brooks, 1998a). Regarding the adaptive signi®cance of the
accumulation trait, an antiherbivore defensive function has
been proposed (Boyd, 1998). Relative to the above is the
question of the distribution of nickel within the plant body.
Leaves seem to be the main sinks of nickel (Severne, 1974;
Mesjasz-Przybylowicz et al., 1994; KraÈmer et al., 1997b); in
the three cases where inter-tissue distribution within a leaf
was studied, nickel was found to be deposited in the
epidermis and its appendages (Severne, 1974; MesjaszPrzybylowicz et al., 1994; KraÈmer et al., 1997a). There have
been even fewer investigations in which the intercellular
* For correspondence. Fax ‡3 061 997411, e-mail g.k.psaras@
upatras.gr
0305-7364/00/070073+06 $35.00/00
distribution in the epidermis has been addressed (Heath
et al., 1997).
In the present investigation, a combination of scanning
electron microscopy and X-ray microanalysis was used to
study the relative abundance of nickel in various epidermal
cell types of eight hyperaccumulating Brassicaceae species
grown on ultrama®c soils in Greece.
M AT E R I A L S A N D M E T H O D S
Plant material
The work presented here was carried out using dry leaves
from plant material kept in the herbarium at the University
of Patras, Greece. Ten species belonging to the family
Brassicaceae grown on ultrama®c soils in Greece were
chosen for the study. Eight of these are known to be metal
hyperaccumulators (Brooks, 1998b), while the other two
non-accumulating species from the serpentine ¯ora were
included for comparison. Plant material was taken from the
following specimens.
Nickel accumulators: Alyssum euboeum HalaÂcsy (388460 N,
238190 E, Phitos & Kamari 20241), Alyssum heldreichii
Hausskn. (398470 N, 218130 E, Charpin et al. AC 11124),
Alyssum lesbiacum (P. Candargy) Rech. ®l. (398020 N,
268180 E, Strid et al. 26184), Alyssum smolikanum NyaÂraÂdy
(408020 N, 218050 E, Phitos et al. 25666), Bornmuellera
baldacii (Degen) Heywood ssp. baldacii (408060 N, 208590 E,
Hartvig & Seberg 4407), Bornmuellera tymphaea (Hausskn.)
Hausskn. (398490 N, 218230 E, Constantinidis 8007), Leptoplax emarginata (Boiss.) O. E. Schulz (398490 N, 218240 E,
Constantinidis 8049) and Thlaspi pindicum Hausskn.
(398070 N, 228120 E, Constantinidis 7394).
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Psaras et al.ÐNickel Localization in Metal Hyperaccumulators
Non-accumulators: Aubrieta glabrescens Turrill (408050 N,
208540 E, Phitos et al. 25579) and Erysimum microstylum
Hausskn. (398090 N, 228040 E, Constantinidis & Iliadis 7812).
To ascertain whether the drying procedure involved in
the preparation of the herbarium specimens could have
caused redistribution of the element, fresh material from the
accumulators T. pindicum and L. emarginata growing wild
on ultrama®c soil (398060 N, 228180 E and 398080 N, 228110 E,
respectively) was also examined. Mature leaves were
harvested, stored in air-tight plastic bags and immediately
transferred to the laboratory for microanalysis.
Microscopy and microanalysis
In preliminary trials, fresh plant material from Thlaspi
pindicum and Leptoplax emarginata was mounted on the
stage of a scanning electron microscope (Jeol 6300 SEM,
Tokyo), immersed in liquid nitrogen within a cryo-transfer
unit (CT 1500 Oxford Instruments, Oxford, UK) and
transferred to the SEM. The plant material was observed at
low voltage before it was slightly etched for a few seconds at
ÿ908C, moved back to the cryo-preparation chamber and
coated with gold. The specimens were examined at 20 kV.
Dry leaves from the same species kept in the University of
Patras Herbarium were examined for comparison. The dry
plant material was mounted on stubs with double-sided
adhesive tape, sputter coated with gold and observed at
room temperature. X-ray maps and energy dispersive
spectra (EDS) derived from both fresh and dry material
showed a similar relative distribution of Ni between the
various epidermal cell types, indicating that the drying
procedure did not cause substantial element redistribution.
Therefore, further work was carried out using dry
herbarium material. Four to ®ve leaves from di€erent
individuals of each species were sampled.
Dry plant material was mounted on stubs with doublesided carbon adhesive tape and sputter coated with gold.
The samples were examined with a Jeol 6300 scanning
electron microscope connected to a Link Pentafet (model
6699) system (Oxford Instruments) for microanalysis. The
system is equipped with a Si(Li) detector and a thin window
(Be-window) for elements from B to U. The accelerating
voltage was 20 kV, the working distance 15 mm and the
probe current 15 nA. During microanalysis the specimen
temperature was about 208C and the column vacuum
3 10 ÿ6 torr. Quantitative calibration was made by means
of standard specimens from MAC (Microanalysis Consultants Ltd). Element mapping, composition and energy
dispersive spectra were processed using a Link ISIS software (series 300, revision 3.2, Oxford), enabling the system
to give directly the elemental composition percentage for
each selected element. Although it is preferable to use
aluminium coating for quantitative estimation of element
concentrations (McCully et al., 1998), quantitative data on
nickel abundance given here are relative, thus are not
a€ected by the gold coating; elemental concentrations were
estimated excluding gold from the normalized percentages.
The signal for each element (including Ni) results from the
corresponding `Hall voltage' level created from the collisions of the photons on the Si(Li) detector. Mapping was
displayed using the SpeedMap software, including CAMEO
(Link ISIS). The colouring during mapping is achieved by a
very high number of point microanalyses made several
times per second. The grid for the points of microanalysis
was set at ultra-®ne resolution. Micrographs and X-ray
maps made on the computer monitor were taken in digital
form.
R E S U LT S
Whole leaf
Examination of leaf transverse sections under the analytical
SEM revealed that nickel accumulates in the epidermis of
both leaf surfaces. An X-ray map from a cross-sectioned
Thlaspi pindicum leaf is shown in Fig. 1A.
Trichomes
Seven of the eight nickel hyperaccumulators studied here
possess either isolated non-glandular hairs or dense
trichomes on their leaves. Only one species, namely Thlaspi
pindicum, does not possess trichomes (Fig. 1A, B). Leptoplax emarginata leaves possess isolated T-shaped hairs
(Fig. 1C) on their lower surface only. Bornmuellera baldacii
ssp. baldacii exhibits several asymmetrical stellate hairs
(Fig. 1E) also on its lower leaf surface. Both surfaces of
Bornmuellera tymphaea leaves are covered by a dense layer
of T-shaped hairs (Fig. 1G). All Alyssum species examined
here possess dense trichomes consisting of stellate hairs on
both leaf surfaces (A. euboeum, Fig. 2A; A. heldreichii,
Fig. 2C; A. lesbiacum, Fig. 2E; and A. smolikanun, Fig. 2G).
As shown in the X-ray maps of the leaf surface of the seven
hairy-leaved metal accumulators, nickel seems to be
excluded from the trichomes (Figs 1C, E, H, 2B, C±E,
G, H).
Other epidermal cell types
As shown in X-ray maps of the epidermis, nickel seems to
be excluded from the guard cells and a more-or-less clear
gradient in its relative abundance is established, peaking
away from stomata (Figs 1, 2). This is especially evident in
the cases of Thlaspi pindicum, Leptoplax emarginata,
Alyssum euboeum and A. heldreichii (Figs 1B, D, 2B, F,
respectively) where the highest levels of nickel were found in
large epidermal cells between stomatal complexes. Thus, the
levels of nickel in the subsidiary cells were intermediate,
while in the case of T. pindicum (Fig. 1B), nickel seems to be
excluded from the subsidiary cells as well.
Besides the X-ray maps, energy dispersive X-ray spectra
were also taken. The case of Leptoplax emarginata is shown
in Fig. 3. Figure 3A refers to the whole area shown in the
micrograph of Fig. 1C. Figure 3B±D refers to the small
areas on the T-shaped hair, the epidermal cell and the
stoma, respectively, indicated by the small circles in Fig. 1C.
The inserted numbers indicate the percent contribution of
nickel in the elemental composition in each case. As
expected, this contribution was low in the guard cell
(0.54 %) and the trichome (0.47 %), increasing almost
Psaras et al.ÐNickel Localization in Metal Hyperaccumulators
75
F I G . 1. SEM micrographs combined with X-ray maps from leaves of four nickel-accumulating species. Cross section (A) and surface view (B) of a
Thlaspi pindicum leaf. Leptoplax emarginata leaf under low (C) and high (D) magni®cation. Low (E) and high (F) magni®cation of a Bornmuellera
baldacii ssp. baldacii leaf. SEM and X-ray map of Bornmuellera tymphaea leaf under low (G) and high (H) magni®cation. Green colouration in the
X-ray maps indicates the presence of nickel. Note the absence of nickel from both the hairs and guard cells. Bars ˆ 50 mm.
76
Psaras et al.ÐNickel Localization in Metal Hyperaccumulators
F I G . 2. SEM micrographs and X-ray maps of leaves of four nickel-accumulating Alyssum species. A, B, A. euboeum; C, D, A. heldreichii; E, F,
A. lesbiacum; G, H, A. smolikanum. Low (A, C, E, G) and high (B, D, F, H) magni®cations are shown. Green colouration in the X-ray maps
indicates the presence of nickel. Note the absence of nickel from both the hairs and guard cells. Bar ˆ 200 mm in G; all other bars ˆ 50 mm.
Psaras et al.ÐNickel Localization in Metal Hyperaccumulators
77
B
A
C
C
Ni: 1.09
Ni: 0.47
Caα
Au
Au
O
O
K
Ni Mg
Cl
Caα
Caβ
K
Ni
Mg
Ni
Caβ
Cl
Ni
C
C
D
C
Ni: 4.42
Au
K Caα
Au
Ni
Cl
O
0
Ni Mg
O
Ni Mg
Caβ
2
4
Ni: 0.54
6
8 keV
0
Cl K Caα
Caβ
2
4
Ni
6
8 keV
F I G . 3. Energy dispersive spectra (EDS) taken from the leaf epidermis of Leptoplax emarginata. A, From the epidermal area shown in Fig. 1C;
B±D, from a hair, an epidermal cell and a guard cell as indicated by the circles in Fig. 1C. The inserted numbers indicate the relative elemental
percent contribution of nickel (gold excluded from the normalized percentage).
nine-fold in the epidermal cell (4.42 %). An intermediate
value (1.09 %) was evident for the larger area of Fig. 1C
(i.e. EDS in Fig. 3A), embracing all cell types. Figure 3 also
shows that trichomes are an ecient sink for extra calcium
(see De Silva et al., 1996). Nickel was not detected by X-ray
microanalysis in the non-metal accumulators (i.e. Aubrieta
glabrescens and Erysimum microstylum; results not shown).
DISCUSSION
The results clearly show a consistent exclusion of nickel
from guard cells in all eight nickel accumulators studied. A
similar result was found for Thlaspi montanum examined by
Heath et al. (1997). Therefore, we may assume that nickel
interferes with normal stomatal function and, accordingly,
guard cells must be protected from high concentrations of
the element. The nature of this interference can be deduced
by considering the mechanisms through which an absorbed
toxic metal is rendered inactive, in relation to the speci®c
functions of guard cells. Successful detoxi®cation probably
requires the formation of a stable organometallic complex
and a physiologically inert cell compartment for permanent
storage (see Brooks, 1998a and literature therein). According to the ligand-based classi®cation of Nieboer and
Richardson (1980), nickel falls within the border line
between classes A and B of metals, forming complexes
with ligands containing both carboxyl and sulphydryl
groups. Indeed, complexes of nickel with citrate and malate
have been proposed as mechanisms for nickel detoxi®cation
(Brooks 1998a). With regards storage, a possible candidate
site could be the vacuole, as shown for cadmium (VoÈgeliLange and Wagner, 1990) and zinc (VaÂzquez et al., 1992).
However, vacuoles of guard cells are not physiologically
inert since they are engaged in a complex ion trac through
the tonoplast membrane, leading to the osmotically driven
changes in cell turgor which mediate stomatal movements
(Willmer and Fricker, 1996). Among the translocated ions,
malate is thought to play a crucial role. Therefore, even a
transient presence of nickel in guard cells could immobilize
malate, whose migration between the cytoplasm and
vacuole is essential. In addition, ion movements across the
plasma and vacuolar membranes in guard cells require
particular pumping properties, which could be perturbed
by the presence of nickel. Indeed, it has been shown that the
in vitro activity of the plasma membrane ATPase was
severely inhibited by nickel (Ros and Picazo, 1990). The
ATP needed to drive the ion movements may result from
photosynthetic activity of guard cells (Willmer and Fricker,
1996). However, nickel is known to suppress photosynthetic
electron ¯ow (Sheoran et al., 1990) and to impair photosynthetic activity by substituting magnesium in the chlorophyll molecule (KuÈpper et al., 1996). Thus, we may assume
that nickel is incompatible not only with the unique ionmovements characterizing the guard cells, but with their
78
Psaras et al.ÐNickel Localization in Metal Hyperaccumulators
ability to photosynthesize as well. Hence, its epidermal
localization should separate it spatially from these functions. We may add here that the absence of Ni from the
mesophyll cells (Severne, 1974; Mesjasz-Przybylowicz et al.,
1994; KraÈmer et al., 1997b; and results from the present
investigation, e.g. Fig. 1A) can be correlated with its
deleterious e€ects on photosynthesis.
The absence of nickel from the trichomes was rather
unexpected in view of the fact that KraÈmer et al. (1997a),
working with Alyssum lesbiacum (which was also tested in
the present study) found a preferential sequestration of the
metal within epidermal trichomes. The reasons for this
discrepancy are not known at present. We may argue,
however, that in all the species studied here, the trichomes
are non-glandular, probably serving an antitranspirant
function, as judged by their density. Such trichomes lose
their protoplasm and die very early during leaf development
(Uphof, 1962) and in certain cases the walls of the lowest
cells are cutinized to prevent apoplasmic water movement
(Fahn, 1986). Apparently, such structures lack the biochemical and mechanical attributes to accumulate nickel,
unless they do so during the earliest phases of leaf development. Further research is needed to elucidate possible
changes in nickel accumulating capacity between various
cell types during leaf development.
We may conclude that, in the epidermis of hyperaccumulators, nickel is sequestered in physiologically more
inert, yet living, cells. The preferential exclusion from guard
cells and trichomes can be explained on the basis of the
constraints imposed by their function and development.
AC K N OW L E D G E M E N T S
Th.C. is a recipient of a Post-Doctoral Scholarship from the
State Scholarship Foundation.
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