CSIRO PUBLISHING
Functional Plant Biology, 2004, 31, 1061–1074
www.publish.csiro.au/journals/fpb
Studies on spatial distribution of nickel in leaves and stems of the metal
hyperaccumulator Stackhousia tryonii using nuclear microprobe
(micro-PIXE) and EDXS techniques
Naveen P. BhatiaA,B,D , Kerry B. WalshA , Ivo OrlicB , Rainer SiegeleB ,
Nanjappa AshwathA and Alan J. M. BakerC
A Primary
Industries Research Centre, School of Biological and Environmental Sciences,
Central Queensland University, Rockhampton, Qld 4702, Australia.
B Environment Division, Australian Nuclear Science and Technology Organisation (ANSTO),
Lucas Heights, NSW 2234, Australia.
C School of Botany, The University of Melbourne, Vic. 3010, Australia.
D Corresponding author. Email: [email protected]
Abstract. Stackhousia tryonii Bailey is one of the three nickel hyperaccumulators reported from Australia. It
is a rare, herbaceous plant that accumulates nickel (Ni) both in leaf and stem tissues. Localisation of Ni in
leaf and stem tissues of S. tryonii was studied using two micro-analytical techniques, energy dispersive X-ray
spectrometry (EDXS) and micro-proton-induced X-ray emission spectrometry (micro-PIXE). Dimethylglyoxime
complexation of Ni was also visualised by bright- and dark-field microscopy, but this technique was considered to
create artefacts in the distribution of Ni. Energy dispersive X-ray spectrometric analysis indicated that guard cells
possessed a lower Ni concentration than epidermal cells, and that epidermal cells and vascular tissue contained higher
levels of Ni than mesophyll, as reported for other Ni hyperaccumulators. The highest Ni concentration was recorded
(PIXE quantitative point analysis) in the epidermal cells and vascular tissue (5400 µg g−1 DW), approximately
double that recorded in palisade cells (2500 µg g−1 DW). However, concentrations were variable within these
tissues, explaining, in part, the similarity between average Ni concentrations of these tissues (as estimated by region
selection mode). Stem tissues showed a similar distribution pattern as leaves, with relatively low Ni concentration in
the pith (central) region. The majority of Ni (73–85% for leaves; 80–92% for stem) was extracted from freeze-dried
sections by water extraction, suggesting that this metal is present in a highly soluble and mobile form in the leaf
and stem tissues of S. tryonii.
Keywords: elemental mapping, metal hyperaccumulation, micro-PIXE, nickel, nuclear microprobe analysis.
Introduction
Accumulation of certain metal(loid)s [arsenic (As), cadmium
(Cd), cobalt (Co), copper (Cu), manganese (Mn), Ni, zinc
(Zn) and lead (Pb)] in plants above threshold concentration
is generally phytotoxic. However, a small group of plants
(also called metallophytes), occurring on metal-enriched
soils, has the capacity to accumulate (one or more of)
these metals in concentrations that are orders of magnitude
higher than in plants that occur on normal soils (Baker
and Brooks 1989). Hyperaccumulation of metals by plants
is an extremely rare phenomenon (exhibited by <0.2% of
angiosperms; Baker et al. 2000). A nickel hyperaccumulator
is defined as a plant with Ni concentrations exceeding
1000 µg g−1 DW (0.1%) in any above-ground tissue
(Reeves 1992).
All the Ni hyperaccumulating plants reported so far are
endemic to soils derived from ultramafic (serpentinite) rocks.
These soils are infertile owing to low concentrations of major
Abbreviations used: ANSTO, Australian Nuclear Science and Technology Organisation; DMG, dimethylglyoxime; EDSX, energy dispersive
X-ray spectrometry; EPXS, electron probe X-ray spectrometry; ICP-OES, inductively coupled plasma-optical emission spectrometry; micro-PIXE,
micro-proton-induced X-ray emission spectrometry; NIST-SRM, National Institute of Standards and Technology-standard reference material;
OM– DAQ, Oxford Microprobe Data Acquisition System; RBS, Rutherford backscattering spectrometry; SEM, scanning electron microscope;
STEM, scanning transmission electron microscope.
© CSIRO 2004
10.1071/FP03192
1445-4408/04/111061
1062
Functional Plant Biology
N. P. Bhatia et al.
plant nutrients (e.g. nitrogen, phosphorus, potassium and
calcium), and elevated concentrations of elements such as,
nickel, chromium, cobalt, magnesium, iron and manganese.
Ultramafic is relatively rare in Australia compared to other
continents, due to a history of tectonic stability since the
Palaeozoic (Davie and Benson 1997), and occur mainly
in New South Wales, Queensland, Victoria and Western
Australia. In Queensland, the three disjunctive outcrops occur
in the northern, central and south-eastern parts of the state.
In central Queensland, serpentinite belt occurs just north of
the Tropic of Capricorn and covers approximately 100 000 ha
in patches between Marlborough in the north and Canoona
and Bondoola in the south (Fig. 1) (Murray 1969; Forster and
Baker 1997).
Stackhousia tryonii is one of the three Ni
hyperaccumulating plants reported from Australia (Severne
1974; Batianoff et al.1990; Batianoff and Specht 1992). It is a
rare, herbaceous plant that exhibits great serpentinic fidelity,
and accumulates Ni both in leaf and stem tissues to levels up
to 41 300 and 7100 µg g−1 DW, respectively (Batianoff et al.
1990; Batianoff and Specht 1992).
An understanding of the physiology of metal
hyperaccumulation requires quantitative studies of the
distribution of metals within plant tissues. Tolerance to and
Middle Is.
PERCY ISLES
South Is.
22 °
SOUTHERN
PACIFIC
OCEAN
BROAD SOUND
SHOALWATER
BAY
St. Lawrence
Great ba
rrier ree
ay
ay
hw
ilw
Ra
ig
Bruce H
f
Marlborough
Mt. Slopeaway
Princhester
23 °
Canoona
Eden Bann
Marble Ridges
Mt. Etna
Bondoola
N
roy
Fitz
10
5 0
10km
Tungamull
ROCKHAMPTON
Riv
er
150 °
Yeppoon
Mt Wheeler
Emu Park
Keppel Sands
Balnagowan
Tropic of capricorn
151°
Fig. 1. Distribution of serpentine rocks (shaded) and soils in central Queensland.
Spatial distribution of Ni in Stackhousia tryonii
Functional Plant Biology
hyperaccumulation of toxic metals by plants presumably
requires formation of organo-metallic complex(es),
associated with organic compounds such as oxygen
donor ligands (e.g. carboxylates), sulphur donor ligands
(e.g. metallothioneins and phytochelatins) or nitrogen
donor ligands (e.g. amino acids) (Baker et al. 2000),
with transport, compartmentalisation and storage of these
complexes within the vacuoles of ‘storage’ cells. These
storage cells may play an ecophysiological role (e.g.
epidermal storage, anti-herbivory or pathogenicity). In
most hyperaccumulating plants, metal concentrations (per
unit dry matter) are greater in leaf than in stem and in
stem than in root tissue (Mesjasz-Przybylowicz et al. 1994,
2001b; Robinson et al. 2003). Metal accumulation in the
epidermal and sub-epidermal cells of the leaf appears to be
1063
a common feature of metal accumulating plants (Table 1).
For example, in Ni hyperaccumulator Senecio coronatus
(Thunb.) Harv., the ratio of Ni (micro-PIXE analysis) in
whole leaf to epidermis was 1 : 3.3 (Mesjasz-Przybylowicz
et al. 1994). Nickel is preferentially localised in the leaf
epidermal cells of Alyssum lesbiacum (Candargy) Rech.F., A.
bertolonii Desv., and Thlaspi goesingense Hálácsy (Küpper
et al. 2001), Senecio anomalochrous Hilliard. (Balkwill
6869J), (Mesjasz-Przybylowicz et al. 2001a), A. euboeum
Hálácsy, A. heldreichii Hausskn., Leptoplax emarginata
(Boiss.) O.E. Schulz, and Thlaspi pindicum Hausskn. (Psaras
et al. 2000), Hybanthus floribundus (Lindley) F.Muell
(Bidwell et al. 2004, Severne 1974) and Thlaspi japonicum
Boiss. (Mizuno et al. 2003); epidermal cells of leaf
midrib and leaf margin of Berkheya coddii Roessler
Table 1. Review of application of microanalytical techniques (PIXE and EDXS) in nickel hyperaccumulating plants (vegetative parts only)
Ni hyperaccumulators
Plant part examined
Technique
Major site of localisation
Reference
Alyssum lesbiacum
Alyssum lesbiacum,
A. bertolonii and
Thlaspi goesingense
Alyssum euboeum,
A. heldreichii,
A. smolikanum,
Bornmuellera baldacii
B. tymphaea,
Leptoplax emarginata,
L. emarginata and
Thlaspi pindicum
Berkheya coddii
leaf
leaf
micro-PIXE
EDXS
epidermis
epidermis
Krämer et al. (1997)
Küpper et al. (2001)
leaf
EDXS
epidermis
Psaras et al. (2000)
leaf
micro-PIXE
epidermis
Berkheya zeyheri subsp.
rehmannii var.
rogersiana
Hybanthus
austrocaledonicus,
H. caledonicus var.
linearifolia,
Psychotria douarrei,
and H. floribundus
Hybanthus floribundus
Senecio anomalochrous
leaf
EDXS
micro-PIXE
epidermis (cuticle)
epidermis
Mesjasz-Przybylowicz et al.
(1998, 2001b)
Robinson et al. (2003)
Mesjasz-Przybylowicz et al.
(1996a)
leaf surface
micro-PIXE
no evidence
of localisation
Kelly et al. (1975)
leaf
leaf, stem, root
EDXS
micro-PIXE
epidermis
epidermis
Bidwell et al. (2004)
Mesjasz-Przybylowicz et al.
(1997c)
Mesjasz-Przybylowicz et al.
(2001a)
Mesjasz-Przybylowicz et al.
(1994)
Przybylowicz et al. (1995)
Mesjasz-Przybylowicz et al.
(1996a, b, 1997a)
Mesjasz-Przybylowicz et al.
(1997b)
Noell and Morris (1996, 1997)
leaf
Senecio coronatus
leaf, stem
epidermis
micro-PIXE
epidermis
leaf
leaf
epidermis
epidermis
leaf, stem
epidermis (leaf, stem)
and cortex (stem)
epidermis, inside and on
the surface of guard cells
subsidiary cells that
surround guard cells
Stackhousia tryonii
leaf
EPXS / EDXS
Thlaspi montanum var.
siskiyouense
leaf
EDXS
Heath et al. (1997)
1064
Functional Plant Biology
(Mesjasz-Przybylowicz et al. 2001b); epidermal subsidiary
cells of Thlaspi montanum var. siskiyouense P. K. Holmgren
(Heath et al. 1997); leaf (epidermal) trichomes of
A. lesbiacum (Candargy) Rech. F. (Krämer et al. 1997);
epidermal cells (Bidwell 2001; Noell and Morris 1996, 1997)
and cuticle of Stackhousia tryonii (Bidwell 2001)
and B. coddii (Robinson et al. 2003). However, Psaras
et al. (2000) studied the relative abundance of Ni in the leaf
epidermis of eight hyperaccumulators and reported that Ni
is excluded from both trichomes and guard cells.
Other metal species show similar patterns of
accumulation. For example, maximum accumulation
of Zn was reported in the vacuoles of epidermal cells
of leaves of the Zn hyperaccumulating plant Thlaspi
caerulescens J.&C.Presl. (Vázquez et al. 1992, 1994;
Küpper et al. 1999; Frey et al. 2000). Similarly, As was
localised predominantly in both adaxial and abaxial
epidermal cells of the hyperaccumulator Pteris vittata L.
(Lombi et al. 2002). Mesophyll cells have also been reported
to possess substantial concentrations of Ni in B. coddii
(Mesjasz-Przybylowicz et al. 2001b); Zn in T. caerulescens
(Frey et al. 2000) and Arabidopsis halleri L. (Zhao et al.
2000); and Zn and Cd in A. halleri (Küpper et al. 2000).
Dimethylglyoxime (DMG) impregnated paper is often
used for quick identification of Ni hyperaccumulator plants
in the wild (Reeves et al. 1996, 1999), and also for
localisation of Ni (Vergnano Gambi 1967; Heath et al.
1997; Boominathan and Doran 2003; Mizuno et al. 2003).
Dimethylglyoxime complexes Ni to yield a magenta-coloured
product. However, it is likely that redistribution of Ni occurs
during the staining process.
The spatial localisation of metals within biological tissues
can be typically quantified using micro-analytical techniques
based on X-ray emission, following irradiation by charged
particles (electrons, protons or heavier particles). MicroPIXE (micro proton-induced X-ray emission spectrometry;
also known as nuclear microprobe) and EDXS (energy
dispersive X-ray spectrometry) simultaneously measure and
map elemental content. Higher analytical sensitivities are
obtainable using micro-PIXE than EDXS (Przybylowicz
et al. 2001). Density correction routines based on Rutherford
backscattering have improved the accuracy of micro-PIXE
analysis relative to EDXS analysis. Our previous work
(Bhatia et al. 2003) confirmed the reliability of micro-PIXE
as a technique for measurement of elements (including
Ni) within biological tissues (fruits of S. tryonii), and the
results were comparable with those obtained using ICP-OES
(inductively coupled plasma-optical emission spectrometry).
The application of micro-PIXE is typically limited by
accessibility to a proton beam facility. Of the 13 publications
reporting application of micro-PIXE technique for studying
localisation of metals in metal-hyperaccumulating plants, 10
represent the work of one group at the National Accelerator
Centre (NAC), Faure, South Africa (Table 1). The Australian
scientific community has access to this technology through
N. P. Bhatia et al.
the Australian Nuclear Science and Technology Organisation
(ANSTO).
Two (conference) reports have considered the distribution
of Ni in S. tryonii. Noell and Morris (1997) employed EDXS
techniques and reported higher concentrations of Ni in the
epidermis than in other leaf tissues of S. tryonii. However,
Bidwell (2001) found that Ni accumulated on the leaf surface
(cuticle) of this species. It was also reported that Ni was also
present in substantial concentrations within guard cells (Noell
and Morris 1997; Bidwell 2001). Neither study considered
the distribution of Ni in stem tissues. The latter point is of
interest as S. tryonii has reduced leaves, with the stem acting
as the primary photosynthetic organ.
In the present investigation, a combination of SEM
(scanning electron microscope)-EDXS and micro-PIXE
techniques were employed to map the spatial distribution
of metals within transversely-cut leaf and stem sections of
S. tryonii. Energy dispersive X-ray spectrometry was
employed as a qualitative tool, while ‘region selection’ and
‘quantitative point analysis’ techniques of Dynamic Analysis
(micro-PIXE) were used for quantitative assessment of
localised Ni within leaf and stem tissue sections of S. tryonii.
Materials and methods
Plant material
Plants of Stackhousia tryonii Bailey (Fig. 2) were asexually propagated
(Bhatia et al. 2002) from herbaceous heel-cuttings collected from
healthy plants growing in the wild on serpentine soil near Canoona
(23◦ 02 08 S, 150◦ 15 70 E; approximately 50 km north-west of
2 cm
Fig. 2. A young Stackhousia tryonii plant growing naturally on a
serpentine soil in central Queensland.
Spatial distribution of Ni in Stackhousia tryonii
Rockhampton, Queensland). Plants were grown in plastic pots (30 cm
diameter) containing serpentine topsoil (200 mm depth). The pots were
maintained in a polyhouse for several months and supplied with a total of
34 mM Ni solution (as NiSO4 .6H2 O) per pot, a fortnight before specimen
collection. Young branches (which grew during pot culture) of S. tryonii
were excised from the centre of the plant. The basal leaves on these
branches were separated from the stem by cutting at the base. The leaves
were further cut into two halves (across the longitudinal axis) using a
sharp razor blade. The stem was cut into 8–10 mm long segments.
Functional Plant Biology
1065
immediately cryo-fixed in liquid nitrogen. Frozen specimens were then
freeze-dried for 24 h in a VirTis Sentry Vaccu-Freeze (The Virtis Co.,
Gardiner, NY) freeze-drier (referred to as unsoaked specimens). One set
of hand-sectioned, freeze-dried leaf and stem specimens was soaked for
4 h in deionised water (with one change) at ambient conditions (referred
to as soaked specimens) before micro-PIXE analysis. Sections were
individually sandwiched between Formvar films prepared by dissolving
Formvar 15 / 95 resin (ProSciTech, Thuringowa, Qld) in 2% ethylene
dichloride as per the procedure described in Bhatia et al. (2003). The
prepared specimens were stored in a desiccator until analysis.
Sample preparation
SEM-EDXS study
Tissue was prepared by three methods.
Chemical fixation and critical-point drying
Leaf segments were soaked overnight in DMG (1% solution in
95% ethanol) at room temperature and fixed in 2.5% glutaraldehyde in
0.1 M phosphate buffer (pH 6.9) overnight in a cold room at 8◦ C. Fixed
specimens were rinsed twice for 2 min in distilled water and dehydrated
in steps with an ascending series of aqueous acetone (10% w / v; each
step for 10 min). Dehydrated specimens were critical point dried in a
Polaron E3000 (Watford, England) using acetone as the intermediate
fluid. Another set of specimens was treated in exactly the same way but
without soaking in DMG. The critical point dried specimens were cut
into halves with a sharp razor blade and were mounted on to grooves cut
into aluminium stubs to expose the cross-sections. After X-ray (EDXS)
analysis, the specimens were gold coated for 10 min in a Bio-Rad
Polaron SC515 SEM coating system (Fisons Instruments, East Sussex,
UK) before photomicrographs of the analysed tissue were taken.
Resin embedding
Leaf segments (cut transversely into two pieces) were chemically
fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 6.9)
overnight in a cold room at 8◦ C. Fixed specimens were rinsed
twice for 2 min in distilled water and dehydrated in steps with an
ascending series of aqueous ethanol (10% w / v; each step for 10 min).
The dehydrated specimens were infiltrated with LR White resin
(ProSciTech, Queensland, Australia) overnight in a cold room at 8◦ C.
The following day, resin-infiltrated leaf segments were embedded in
resin mixed with a drop of activator in Teflon moulds for 24 h at room
temperature. Leaf sections were cut using a disposable stainless steel
knife fitted on a microtome (Leica Microsystems Pty Ltd, North Ryde,
NSW).
The sectioned specimens were dyed with toluidine blue on a glass
slide, heat-fixed and covered with coverslips using a resin mountant
(DPX; distrene, plasticiser, xylene) before being examined under a light
microscope (Eclipse E 600, Nikon, Tokyo, Japan).
Freeze-fracturing
Leaves of S. tryonii growing in pots were detached from the plant and
immediately plunged into liquid nitrogen. Frozen samples were placed
in grooves cut into aluminium stubs that were pre-cooled with liquid
nitrogen. Prior to mounting the aluminium stubs on a frame, the leaf
samples were snapped (freeze fractured), using pre-cooled tweezers.
This procedure exposed a cross section of leaf tissues. The frame was
then placed in the vacuum chamber of the SEM. Since the SEM used
was not fitted with a cryostage, elemental mapping was restricted to
the time until which the samples warmed to melting point, which was
typically a few minutes.
Micro-PIXE study
For micro-PIXE analysis, the sample preparation protocol established
by Przybylowicz et al. (1999) was followed with some modifications.
Fresh leaf and stem samples were hand-sectioned with a razor blade and
Instrumentation and analytical methods
SEM-EDXS study
Microanalysis of leaf and stem surface and cross-sections was performed
in a JEOL JSM-5300 LV scanning electron microscope (JEOL USA Inc.,
MA) attached to a Link PentaFET system (Oxford Instruments, Oxford,
UK) interfaced with Link eXII Analysis software. X-rays were detected
by a Si(Li) detector with a thin beryllium window. The microscope
was operated at an acceleration voltage of 15 kV with a collection time
of 100 s. Working distance was maintained at 15 mm and stage-tilt was
adjusted to obtain a take-off angle of 40◦ . Energy dispersive spectra (and
images) were processed using Link eXII Analysis software. The SEMEDXS setup was calibrated using a cobalt reference standard (Astimex
Scientific Ltd, Toronto, Canada), with elemental composition reported
as a normalised percentage (dry) weight basis of the total detectable
elements (Z > 9) (note that this is a limitation of the EDXS unit used,
other systems may have lower detection limits). As biological material is
primarily composed of non-detectable C, H, O and N, it is not surprising
that the EDXS assessed elemental concentrations were not in agreement
with results obtained using micro-PIXE or ICP-OES (see Bhatia
et al. 2003). As such, the EDXS result is not accurate. However, they
were relatively precise, with repeatable results possible for a given
location. Therefore the SEM-EDXS measurements were considered to
be qualitative only.
Micro-PIXE study
A 10 MV Tandem accelerator at ANSTO provided a 3 MeV
proton beam for the nuclear microprobe analyses. For point
analysis and dynamic elemental mapping, both PIXE and
proton backscattering (BS) were employed simultaneously.
Details of PIXE set up, standardisation techniques and data
acquisition procedures have been described in detail in Bhatia et al.
(2003). Average micro-PIXE analyses were shown to be consistent
with ICP analysis.
The PIXE spectra were analysed using GeoPIXE II software
(for a discussion of the PIXE technique, see Witkowski et al.
1997). This program allows extraction of PIXE elemental maps
from the accumulated data, using Dynamic Analysis (Ryan 2001),
which are overlap-resolved and background-subtracted. Using
the region selection mode (Dynamic Analysis), various regions
(epidermis, mesophyll, vascular tissue, cortex, pith) from the images
[reproduced on computer screen from data accumulated by the
Oxford Microprobe Data Acquisition System, OM– DAQ, Grime
and Dawson 1995)] of transversely cut leaf and stem sections were
selected. Region selection was done by encircling the anatomical
areas of interest, using the drawing mode of the Dynamic Analysis.
Owing to the low resolution of acquired elemental maps, region
selection for ‘epidermis’ included epidermal cell wall, cuticle and
sub-epidermal tissues. Alternatively, the quantitative point analysis
feature of Dynamic Analysis was employed using the same datasets
for transect(s) across the whole specimen. Transects were located
on the basis of visual inspection of the elemental distribution
maps.
1066
Functional Plant Biology
N. P. Bhatia et al.
Results
Light- and bright-field microscopy
Leaf and stem anatomy
Leaves were composed of 3–4 layers of palisade mesophyll
above spongy mesophyll cells (Fig. 3a). The epidermal cells
on the adaxial portion of the leaf were larger than those on
the abaxial side. Stomata were present at similar densities
(data not shown) on both adaxial and abaxial sides
of leaf material. Leaf vasculature was dominated by
a central vein surrounded by a bundle sheath layer
(Fig. 3a). Spherical to elliptical chloroplasts were observed
in the mesophyll cells at higher magnification (data not
shown).
a
E
P
VB
S
M
100 µm
b
c
100 µm
20 µm
Fig. 3. Photomicrograph (light microscope) of a transversely-cut (10 µm) resin-embedded section of Stackhousia
tryonii leaf, (a) stained with toluidine blue. E, epidermis; S, stomata; P, palisade mesophyll; VB, vascular bundle;
M, spongy mesophyll, (b) a dark-field image of a leaf section soaked in dimethylglyoxime for 12 h. Bright white
areas within palisade mesophyll, spongy mesophyll and vascular tissues represent conjugated nickel crystals, and
(c) a bright-field image of a leaf showing conjugated nickel crystals in the vascular bundles and parenchyma cells
(around vascular bundles).
Spatial distribution of Ni in Stackhousia tryonii
The stem comprised of a concentric ring of vascular tissue
separating pith from a photosynthetic cortex. Clusters of thick
walled sclerenchymatous cells occurred at between six and
nine points around the outer cortex of the stem (Fig. 4a).
Functional Plant Biology
1067
white’ areas within leaf sections (Fig. 3b). The conjugated
crystals (Fig. 3c) were verified to contain Ni (approximately
18%) by EDXS spot analysis (data not shown).
SEM-EDXS study
DMG complexation of nickel
In DMG-treated sections, large numbers of magenta
coloured (conjugated Ni) crystals were noted using
bright-field microscopy throughout the leaf and stem, with an
apparent concentration in the inner palisade and mesophyll
and vascular tissues (data not presented). These crystals were
also distinguishable, using dark-field microscopy, as ‘bright
In non-DMG-treated, freeze fractured material, Ni levels
were apparently 100% higher in epidermal cells than
in guard cells. Assessed nickel concentrations were also
(approximately 20–30%) lower within deeper layers of
palisade or mesophyll cells, relative to epidermal cells (data
not shown). Xylem tissue also had lower Ni concentration
than epidermal cells (data not shown).
b
a
c
d
250 µm
Fig. 4. Typical secondary electron image (a) and distribution patterns of calcium (b), potassium (c) and nickel (d) in
transversely-cut, unsoaked stem sections of Stackhousia tryanii (micro-PIXE analysis). Stems were hand sectioned, cryofixed and freeze dried before micro-PIXE analysis. Transverse on nickel map relates to quantitative point analysis across
stem section shown in Fig. 5. Colour intensity denotes the concentration of elements (yellow, high; orange, low).
1068
Functional Plant Biology
N. P. Bhatia et al.
Micro-PIXE study
Leaf sections
Stem sections
Micro-PIXE analysis of unsoaked stem sections revealed
the concentration of three major elements to follow the
order K > Ca > Ni (Table 2; Figs 4, 5). The soaked stem
sections lost nearly all K and approximately 88% of Ni
(Table 2).
Quantitative point analysis (Fig. 5) of the stem
sections demonstrated that Ni concentration varied between
500 µg g−1 DW in the pith region and up to 3000 µg g−1
DW in the epidermal and vascular tissues. The cortex
possessed an intermediate Ni concentration between 800 and
2000 µg g−1 DW. The Ni ‘tissue average’, determined using
the region selection mode of Dynamic Analysis (Table 2), of
the three anatomical regions, epidermal tissues, mesophyll
and vascular tissues of stem, was similar (between 903 and
1067 µg g−1 DW). The pith contained only 344 µg Ni g−1
DW.
The highest concentration of K (region selection mode
of Dynamic Analysis) was measured within vascular tissues
(5533 µg g−1 DW), whereas epidermal tissues contained
4167 µg g−1 DW K. Maximum Ca (region selection) was
localised within epidermal tissues (2733 µg g−1 DW), and
vascular tissues contained intermediate concentrations at
2067 µg g−1 DW.
The concentrations of all elements (including trace
elements) were found to be the lowest within pith (Fig. 5).
Concentrations of K and Ca were nearly half the maximum
concentrations, whereas those of Ni were only one-third of
the maximum.
The concentrations of trace elements (Fe, Zn and Mn)
decreased from the epidermis, through mesophyll to vascular
tissues.
Micro-PIXE analysis detected the presence of three
major elements in the unsoaked leaves of S. tryonii,
with concentration following the order: Ca > K > Ni and
Fe > Mn > Zn (Table 3).
A peak Ni concentration of 5400 µg g−1 DW was recorded
in the epidermis and vascular tissues, compared with 2500
and 3800 µg Ni g−1 DW in the palisade and mesophyll
tissues, respectively (quantitative point analysis; Fig. 7).
Concentrations, however, were variable within tissues,
such that when averaged across anatomical regions, little
difference in Ni concentration between tissues (2400, 2000
and 2600 µg g−1 DW, in epidermal cells, mesophyll cells and
vascular tissue, respectively; region selection; Table 3) was
noted.
Potassium accumulation followed similar pattern of spatial
distribution as Ni (quantitative point analysis; Fig. 7),
reaching a maximum in vascular tissues (up to 7800 µg g−1
DW), followed by epidermal tissues (up to 6000 µg g−1
DW), while palisade and mesophyll tissues possessed nearly
half the concentration (4000 µg g−1 DW) that of vascular
tissues. Again, concentrations were variable within each
tissue, such that average K concentrations across individual
tissues were 2750, 2900 and 3950 µg g−1 DW for epidermal
tissues, mesophyll and vascular tissues, respectively (region
selection; Table 3).
Quantitative point analysis revealed that Ca localisation
also followed a similar trend, with maximum accumulation
in the epidermal tissues (up to 1.5 × 104 µg g−1 DW),
while vascular tissues and mesophyll possessed 1.3 × 104
and 5.0 × 103 µg g−1 Ca DW, respectively (Fig. 7). Again,
average concentration across individual tissues (region
selection; Dynamic Analysis) suggested that Ca was
Table 2. Average concentrations of elements within stem sections of Stackhousia
tryonii as determined by micro-PIXE analysis
Values are mean ± standard error for whole section, epidermal tissue, mesophyll and
vascular tissue for three replicate stem sections; for pith, values are mean, n = 2. MicroPIXE results were obtained using region selection mode of Dynamic Analysis. Percent
loss was calculated from the elemental content of a section subjected to water soaking for
4 h and the average concentration of unsoaked sections (bottom two rows)
Element
Whole section
Concentration (µg g−1 DW)
Epidermal tissue
Mesophyll
Vascular tissue
Pith
K
Ca
Ni
Fe
Mn
Zn
4367 ± 88
2100 ± 100
916 ± 38
156 ± 48
156 ± 1
36 ± 4
4167 ± 120
2733 ± 176
1067 ± 33
303 ± 118
185 ± 3
50 ± 4
3967 ± 233
1633 ± 67
1001 ± 114
133 ± 22
158 ± 11
44 ± 6
5533 ± 285
2067 ± 120
903 ± 24
59 ± 3
155 ± 1
29 ± 4
2600
1200
344
58
67
7
K
Ni
99
88
99
86
99
92
98
80
99
92
Spatial distribution of Ni in Stackhousia tryonii
1 × 105
8 × 104
E
M
VB
P
Functional Plant Biology
VB
predominantly localised in epidermal tissues (4550 µg g−1
DW) and within vascular tissue (4600 µg g−1 DW), while
mesophyll contained nearly two-thirds the concentration
(3050 µg Ca g−1 DW) of epidermal and vascular tissue.
The concentrations of trace elements such as,
Fe, Zn and Mn were 123, 4 and 57 µg g−1 DW,
respectively in the whole leaf section (region selection;
Table 3).
In contrast to unsoaked specimens, the soaked leaf
specimens contained very low K and Ni. The maximum
percent loss of K and Ni was up to 91% and 85%, respectively
from within vascular tissues, and up to 86% and 73%,
respectively, from epidermal tissues. Mesophyll tissue lost
intermediate levels of K (90%) and Ni (78%) (Table 3).
M E
Cl
6 × 104
4 × 104
2 × 104
2.0 × 105
K
Concentration (µg g–1 dry weight)
1.5 × 105
1.0 × 105
5.0 × 104
0
8 × 104
Ca
6 × 104
4 × 104
2 × 104
0
2.0 × 104
Discussion
Fe
Technique
1.5 × 104
104
1.0 ×
5.0 × 103
0
4 × 104
Ni
3 × 104
2 × 104
1 × 104
0
1069
0
400
800
1200
1600
Microns
Fig. 5. Quantitative point analysis (micro-PIXE) across the transect
outlined in Fig. 4. E, epidermis; M, mesophyll; VB, vascular bundle;
P, pith.
Dimethylglyoxime (DMG) has frequently been used as a
histochemical stain for localisation of Ni within tissues of
hyperaccumulator plants (Vergnano Gambi 1967; Severne
1974; Farago and Mahmoud 1983; Heath et al. 1997; Mizuno
et al. 2003). However, it is likely that movement of Ni on
the solvent front occurred during DMG staining, resulting
in redistribution and localisation of conjugated Ni crystals
in the palisade and spongy mesophyll and vascular tissues
(Fig. 3b). Other artefacts may also occur (e.g. pH dependent
staining). To avoid redistribution or movement of Ni on
the solvent front, tissue should be either cryo-substituted
(in DMG solution) at ultra-low temperatures, or alternative
procedures should be considered.
Table 3. Average concentrations of elements within leaf sections of Stackhousia
tryonii as determined by micro-PIXE analysis
Values represent the mean of two replicate leaf sections for whole section, epidermal
tissue, mesophyll and vascular tissue [the difference between replicates (whole
section) was 6, 19, 9, 45, 61 and 16% for K, Ca, Ni, Fe, Zn and Mn, respectively].
Micro-PIXE results were obtained using region selection mode of Dynamic Analysis.
Percent loss was calculated from the elemental content of two replicate leaf sections
subjected to water soaking for 4 h and average concentration of unsoaked sections
(bottom two rows)
Element
Whole leaf section
Concentration (µg g−1 DW)
Epidermal tissue Mesophyll
Vascular tissue
K
Ca
Ni
Fe
Mn
Zn
3000
3900
2200
123
57
4
2750
4550
2400
206
61
11
2900
3050
2000
63
50
<4
3950
4600
2600
54
72
5
K
Ni
89
79
86
73
90
78
91
85
1070
Functional Plant Biology
SEM-EDXS is a versatile technique for estimating the
spatial distribution of chemical elements in cells (Zierold
and Hagler 1989), and has been extensively used for
localisation (and quantification) of heavy metals in various
hyperaccumulating taxa (Heath et al. 1997; Psaras et al.
2000; Küpper et al. 2001; Robinson et al. 2003). However,
it suffers from poor quantification because of the difficulty
involved in estimating the volume of the tissue sampled.
Freeze-fractured material used in the current study would
have freeze-dried during irradiation. This reduces the spatial
resolution of the analysis, since the electron beam penetrates
much deeper into dried compared to freeze-dried tissues.
Furthermore, the instrumentation employed did not allow for
use of oxygen (of water from freeze-fractured specimens) as
an internal standard. Thus, in the current study, the SEMEDXS instrumentation and protocol employed allowed only
qualitative analysis of Ni. Therefore, the discussion is limited
to cellular / tissue-level observations. As noted in a previous
study (Bhatia et al. 2003), the methodology / instrumentation
employed in the current SEM-EDXS study allowed for
precision, but lacked accuracy (data not shown).
The micro-PIXE technique, using Rutherford
backscattering correction, potentially allows quantitative
analysis, but has relatively lower spatial resolution. However,
micrometer level resolution has been reported (Malmqvist
1995). With the nuclear microprobe (micro-PIXE), elemental
maps (Ni, K and Ca) were prepared with a beam resolution
(pixel size) of 2 × 2 µm. Effective resolution would be much
less, given variations in specimen density and movement in
the sample during irradiation.
Witkowski et al. (1997) noted that data from quantitative
point analysis is more reliable than that from region selection
analysis, noting five- and 10-fold difference between region
selection analysis and point analysis for barium and cobalt
content of dormant seeds of Burkea africana. Region
selection analysis was suggested to be limited by the use
of approximations rather than full non-linear least-square
fit to point X-ray spectra (Witkowski et al. 1997). On
the contrary, in a more recent publication, Przybylowicz
et al. (2001) suggested that ‘owing to the heterogeneity of
biological specimens, point analysis may sometimes not
be representative’. We consider that the two methods are
complimentary in that the region selection allows averaging
across a region with loss of specific spatial resolution,
while point analysis allows more accurate estimation
of specific values of elemental / metal composition. For
example, point analysis transects across transverse sections
recorded substantial differences in concentrations within an
identified tissue (particularly epidermal and vascular tissues)
(Fig. 7), as did elemental distribution maps (Fig. 6).
Stackhousia tryonii is a nickel hyperaccumulator
This is the first report on the application of micro-PIXE
technique for localisation of Ni in both the leaf and stem
N. P. Bhatia et al.
a
b
c
100 µm
Fig. 6. Elemental maps of calcium (a), potassium (b) and nickel
(c) within transversely-cut unsoaked leaf sections of Stackhousia tryanii
(mirco-PIXE analysis). Leaves were hand sectioned, cryo-fixed and
freeze dried before micro-PIXE analysis. Transverse on nickel image
related to quantitative point analysis across leaf section shown in Fig. 7.
Colour intensity denotes the concentration of elements (yellow, high;
orange, low).
Spatial distribution of Ni in Stackhousia tryonii
Concentration (µg g–1 dry weight)
E
P
VB
Functional Plant Biology
M
The epidermis as a major storage site for Ni
E
2000
1500
1000
500
0
8000
6000
4000
2000
0
1.5 × 104
Cl
K
Ca
1.0 × 104
5.0 × 103
0
250
200
150
100
50
0
6000
Co
Ni
4000
2000
0
0
115
230
345
460
Microns
575
1071
690
Fig. 7. Quantitative point analysis (micro-PIXE) across the transect
outlined in Fig. 6. E, epidermis; P, palisade mesophyll; M, spongy
mesophyll; VB, vascular bundle.
tissues of S. tryonii. The results confirmed the presence
of the Ni hyperaccumulation trait (>1000 µg Ni g−1 DW;
Reeves 1992) in both the leaf and stem tissues of S. tryonii,
as previously reported for plants grown in a glasshouse
(6900 µg Ni g−1 leaf DW, Bidwell 2001) or occurring in the
natural habitats (up to 41 300 µg Ni g−1 leaf DW, Batianoff
and Specht 1992) and those preserved in the herbaria (up to
21 500 µg Ni g−1 leaf DW and up to 7100 µg Ni g−1 stem
DW; Batianoff et al. 1990), analysed using ICP-based
techniques.
In the present study, the concentrations of
hyperaccumulated Ni in leaf sections were almost double
those found in the stem sections. These results are consistent
with those of Batianoff et al. (1990). As evidenced by the
natural occurrence of various populations on serpentine soils
(most with elevated levels of available Ni; Bhatia 2003), this
species is capable of tolerating otherwise phytotoxic levels
of available Ni. It can therefore be concluded that S. tryonii
is an absolute metallophyte (Baker 1981), and its tolerance
to and hyperaccumulation of Ni are constitutive properties,
as would be expected in such species.
Energy Dispersive X-ray Spectrometric and micro-PIXE
analyses demonstrated the presence of elevated Ni
concentrations within epidermal tissue (cuticle, epidermal
and sub-epidermal cells), with concentrations decreasing
in the deeper layers of palisade and mesophyll cells. Our
results are consistent with previously reported studies on Ni
hyperaccumulating taxa (for examples, see Table 1).
The absolute Ni distribution within leaf tissues, however,
will be a function of Ni concentration within tissue and the
tissue volume. Further information (on epidermal, mesophyll
and vascular volume within the leaf) is required to resolve
this point in S. tryonii. In addition, further information is
also required to explain the spatial variability in Ni content
observed within a tissue type (particularly epidermal tissue).
For example, Küpper et al. (1999) demonstrated a correlation
between cell size and zinc concentration in the epidermis of
Thlaspi caerulescens, while Frey et al. (2000) found that this
correlation was caused by a functional differentiation of the
epidermal cells (accumulation of zinc was found to occur
mainly in the vacuoles of large elongated cells).
Cuticle and guard cells
Bidwell (2001) noted the cuticle to be the predominant
site for Ni accumulation (2078 µg g−1 v. 536 µg g−1 within
epidermal cells of embedded leaf tissue) in S. tryonii. In
the present study, estimation of Ni concentrations within the
cuticle was not possible due to comparatively lower resolution
of the instrumentation employed. However, Ni accumulation
on the outer surface of the epidermis was not visualised
in SEM (backscatter mode), and no abnormal accumulation
of Ni was quantified using micro-PIXE, which would have
been apparent in the outer tissues in the elemental map
(Fig. 6c) or in the quantitative traverse point analysis across
the transect (Fig. 7) of the specimens examined. Contrary
to the observation of Bidwell (2001), Ni was localised in
the sub-epidermal tissues (Fig. 6c), as evidenced in both the
elemental map and the point analysis. Further, it is difficult
to postulate a mechanism by which Ni could accumulate
in the cuticle, given the lack of excretory structures in
the epidermis. However, recently, excretion of Ni through
guttation fluid in Ni hyperaccumulator T. japonicum has also
been demonstrated (Mizuno et al. 2003).
Noell and Morris (1997) observed a high atomic number
deposit on the surface of the epidermis of S. tryonii leaves,
and suggested that Ni containing solutes had moved to the
leaf surface through stomata and wounds resulting from
insect herbivory. This feature may have been an artefact of
sample preparation technique (Noell and Morris 1997), as
specimens were pressed while drying before carbon-coating
for examination using scanning electron microscope. This
application of pressure may have fractured epidermal cells,
resulting in leakage and accumulation of Ni-rich cell contents
on the leaf surface.
1072
Functional Plant Biology
In general, guard cells (stomates) of metal
hyperaccumulating plants are not preferred sites for metal
accumulation (Heath et al. 1997; Psaras et al. 2000) and
accumulate relatively smaller amounts of metals compared
to other surrounding epidermal cells. For example, in
T. caerulescens, guard cells contained less than 2% of the Zn
concentration noted within vacuoles of epidermal cells (Frey
et al. 2000). However, in our study, substantial amounts of Ni
(approximately 50% of the epidermal cells; EDXS study, data
not shown) were noted within the guard cells of S. tryonii leaf
specimens. This relatively high measured Ni concentration
in the guard cells may be an artefact of the limited spatial
resolution of the measuring technique employed. However,
our results are in general agreement with those of Bidwell
(2001) who used scanning transmission electron microscopy
(STEM) of thin sections, noting similar Ni concentrations
within guard (7.5 ± 4 mmol k g−1 embedded tissue) and
epidermal cells (9.1 ± 1.2 mmol k g−1 embedded tissue).
Form of hyperaccumulated Ni
A loss of 73–85% Ni from soaked leaf and 80–92% from
soaked stem specimens suggests that a major portion of Ni
within leaf and stem tissues was present in water-soluble
form. Our results corroborate with those of Bidwell (2001),
who also reported that up to 72% Ni in S. tryonii stems
was water-soluble (no values for leaf tissue were reported).
Citric (Bidwell 2001) and malic acids (Bhatia 2003) have
been implicated to be the major organic ligands possibly
involved in supporting detoxification / transport and storage
of Ni in S. tryonii. Presumably, remaining Ni was bound to
the cell wall material of the tissues, as reported for other Ni
hyperaccumulators T. goesingense (Krämer et al. 2000) and
H. floribundus (Bidwell et al. 2004).
Ecophysiological roles for Ni
Several hypotheses have been proposed to account
for the ecological and evolutionary significance
of the hyperaccumulation trait (Boyd and Martens
1992). A preferential localisation of Ni in the plant
epidermis may be of significance for plant defence against
insect herbivory (Schoonhoven et al. 1998), and might be
toxic to pathogens, given the use of metal compounds (in
particular copper and nickel) as fungicides and bactericides
(Boyd and Martens 1992). However, clear evidence is
lacking to support the hypothesis that hyperaccumulated Ni
confers advantage to S. tryonii plants against herbivory or
pathogenic fungi / bacteria.
Acknowledgments
A merit scholarship (UPRA) to NPB by the Central
Queensland University and financial assistance (Grant No.
450; 02 / 001P) for the present work by the Australian Institute
of Nuclear Science and Engineering (AINSE) is gratefully
acknowledged. We also acknowledge the technical assistance
N. P. Bhatia et al.
of Mr Barry Hood (laboratory work) and Mr V. McCafferty
(SEM work), and the assistance of Queensland Parks and
Wildlife Services (Central Region) for granting a scientific
permit to collect S. tryonii material from its natural habitat.
We thank two anonymous reviewers for their constructive
comments, incorporated into the final text.
References
Baker AJM (1981) Accumulators and excluders — strategies in the
response of plants to heavy metals. Journal of Plant Nutrition 3,
643–654.
Baker AJM, Brooks RR (1989) Terrestrial higher plants which
hyperaccumulate metallic elements — a review of their distribution,
ecology and phytochemistry. Biorecovery 1, 81–126.
Baker AJM, McGrath SP, Reeves RD, Smith JAC (2000) Metal
hyperaccumulator plants: a review of the ecology and physiology
of a biological resource for phytoremediation of metal-polluted
soils. In ‘Phytoremediation of contaminated soil and water’.
(Eds N Terry, G Bañuelos) pp. 85–107. (CRC Press: Boca
Raton)
Batianoff GN, Specht RL (1992) Queensland (Australia) serpentinite
vegetation. In ‘The vegetation of ultramafic (serpentine) soils’.
Proceedings of the first international conference on serpentine
ecology. (Eds AJM Baker, J Proctor, RD Reeves) pp. 109–128.
(Intercept Ltd: Andover)
Batianoff GN, Reeves RD, Specht RL (1990) Stackhousia tryonii Bailey:
a nickel-accumulating serpentine-endemic species of Central
Queensland. Australian Journal of Botany 38, 121–130.
Bhatia NP (2003) Ecophysiology of nickel hyperaccumulation in
Stackhousia tryonii Bailey. PhD thesis, Central Queensland
University, Australia.
Bhatia NP, Bhatia P, Ashwath N (2002) Asexual propagation of
Stackhousia tryonii: a step towards restoration of a rare metallophyte.
Australian Journal of Botany 50, 577–582. doi: 10.1071/BT01035
Bhatia NP, Orlic I, Siegele R, Ashwath N, Baker AJM, Walsh KB
(2003) Elemental mapping using PIXE shows the main pathway
of nickel movement is principally symplastic within the fruit of
the hyperaccumulator Stackhousia tryonii. New Phytologist 160,
479–488. doi: 10.1046/J.1469-8137.2003.00912.X
Bidwell SD (2001) Hyperaccumulation of metals in Australian
native plants. PhD thesis, The University of Melbourne,
Australia.
Bidwell SD, Crawford SA, Woodrow IE, Sommer-Knudsen J,
Marshall AT (2004) Sub-cellular localization of Ni in the
hyperaccumulator, Hybanthus floribundus (Lindley) F.Muell. Plant,
Cell and Environment 27, 705–716. doi: 10.1111/J.0016-8025.
2003.01170.X
Boominathan R, Doran PM (2003) Organic acid complexation, heavy
metal distribution and the effect of ATPase inhibition in hairy roots
of hyperaccumulator plant species. Journal of Biotechnology 101,
131–146. doi: 10.1016/S0168-1656(02)00320-6
Boyd RS, Martens SN (1992) The raison d’Être for metal
hyperaccumulation by plants. In ‘The vegetation of ultramafic
(serpentine) soils’. Proceedings of the first international conference
on serpentine ecology. (Eds AJM Baker, J Proctor, RD Reeves)
pp. 279–290. (Intercept Ltd: Andover)
Davie H, Benson JS (1997) The serpentine vegetation of the WokoGlenrock region, New South Wales, Australia. In ‘The ecology
of ultramafic and metalliferous areas’. Proceedings of the second
international conference on serpentine ecology. (Eds T Jaffré,
RD Reeves, T Becquer) pp. 155–162. (ORSTOM: Nouméa, New
Caledonia)
Spatial distribution of Ni in Stackhousia tryonii
Farago ME, Mahmoud IEDAW (1983) Plants that accumulate metals.
(Part VI): Further studies of an Australian nickel accumulating
plant. Minerals and the Environment 5, 113–121.
Forster BA, Baker DE (1997) Characterisation of the serpentinite
soils of central Queensland, Australia. In ‘The ecology of
ultramafic and metalliferous areas’. Proceedings of the second
international conference on serpentine ecology. (Eds T Jaffré,
RD Reeves, T Becquer) pp. 27–37. (ORSTOM: Nouméa,
New Caledonia)
Frey B, Keller C, Zierold K, Schulin R (2000) Distribution of Zn in
functionally different leaf epidermal cells of the hyperaccumulator
Thlaspi caerulescens. Plant, Cell and Environment 23, 675–687.
doi: 10.1046/J.1365-3040.2000.00590.X
Grime GW, Dawson M (1995) Recent developments in data acquisition
and processing on the Oxford scanning proton microprobe.
Nuclear Instruments and Methods in Physics Research. Section B,
Beam Interactions with Materials and Atoms 104, 107–113.
doi: 10.1016/0168-583X(95)00401-7
Heath SM, Southworth D, d’allura JA (1997) Localization of nickel
in epidermal subsidiary cells of leaves of Thlaspi montanum
var. siskiyouense (Brassicaceae) using energy-dispersive X-ray
microanalysis. International Journal of Plant Sciences 158,
184–188. doi: 10.1086/297429
Kelly PC, Brooks RR, Dilli S (1975) Preliminary observations on the
ecology and plant chemistry of some nickel-accumulating plants
in New Caledonia. Proceedings of the Royal Society of London.
Section B 189, 69–80.
Krämer U, Cotter Howells JD, Charnock JM, Baker AJM, Smith JAC
(1996) Free histidine as a metal chelators in plants that accumulate
nickel. Nature 379, 635–638. doi: 10.1038/379635A0
Krämer U, Grime GW, Smith JAC, Hawes CR, Baker AJM (1997)
Micro-PIXE as a technique for studying nickel localization
in leaves of the hyperaccumulator plant Alyssum lesbiacum.
Nuclear Instruments and Methods in Physics Research. Section
B, Beam Interactions with Materials and Atoms 130, 346–350.
doi: 10.1016/S0168-583X(97)00368-6
Krämer U, Pickering IJ, Prince RC, Raskin I, Salt DE (2000) Subcellular
localization and speciation of nickel in hyperaccumulator and nonaccumulator Thlaspi species. Plant Physiology 122, 1343–1353.
doi: 10.1104/PP.122.4.1343
Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Cellular
compartmentation of cadmium and zinc in relation to other elements
in the hyperaccumulator Arabidopsis halleri. Planta 212, 75–84.
doi: 10.1007/S004250000366
Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP (2001)
Cellular compartmentation of nickel in the hyperaccumulators
Alyssum lesbiacum, Alyssum bertolonii and Thlaspi
goesingense. Journal of Experimental Botany 52, 2291–2300.
doi: 10.1093/JEXBOT/52.365.2291
Küpper H, Zhao FJ, McGrath SP (1999) Cellular compartmentation of
zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant
Physiology 119, 305–311. doi: 10.1104/PP.119.1.305
Lombi E, Zhao FJ, Fuhrmann M, Ma LQ, McGrath SP (2002) Arsenic
distribution and speciation in the fronds of the hyperaccumulator
Pteris vittata. New Phytologist 156, 195–203. doi: 10.1046/
J.1469-8137.2002.00512.X
Malmqvist KG (1995) Biological and Medical Application. In ‘Particleinduced X-ray emission spectrometry, chemical analysis series.
Volume 133’. (Eds SAE Johansson, JL Campbell, KG Malmqvist)
pp. 167–236. (John Wiley and Sons, Inc: New York)
Mesjasz-Przybylowicz J, Balkwill K, Przybylowicz WJ, Annegarn HJ
(1994) Proton microprobe and X-ray fluorescence investigations
of nickel distribution in serpentine flora from South Africa.
Nuclear Instruments and Methods in Physics Research. Section B,
Functional Plant Biology
1073
Beam Interactions with Materials and Atoms 89, 208–212.
doi: 10.1016/0168-583X(94)95174-8
Mesjasz-Przybylowicz J, Balkwill K, Przybylowicz WJ, Annegarn HJ,
Rama DBK (1996a). Similarity of nickel distribution in leaf
tissue of two distantly related hyperaccumulating species. In ‘The
Biodiversity of African plants’. (Eds LJG van der Maesen, XM van
der Burgt, JM van Medenbach de Rooy) pp. 331–335. (Kluwer
Academic Publishers: Dordrecht, The Netherlands)
Mesjasz-Przybylowicz J, Przybylowicz WJ, Prozesky VM, Pineda CA
(1996b) Elemental distribution in a leaf of Senecio coronatus.
Proceedings of the Microscopy Society of South Africa 26, 68.
Mesjasz-Przybylowicz J, Przybylowicz WJ, Prozesky VM (1997a)
Nuclear microprobe investigation of Ni distribution in organs
and cells of hyperaccumulating plants. In ‘The ecology of
ultramafic and metalliferous areas’. Proceedings of the second
international conference on serpentine ecology. (Eds T Jaffré,
RD Reeves, T Becquer) pp. 223–224. (ORSTOM: Nouméa,
New Caledonia)
Mesjasz-Przybylowicz J, Przybylowicz WJ, Prozesky VM, Pineda CA
(1997b) Quantitative micro-PIXE comparison of elemental
distribution in Ni-hyperaccumulating and non-accumulating
genotypes of Senecio coronatus. Nuclear Instruments and Methods
in Physics Research. Section B, Beam Interactions with Materials
and Atoms 130, 368–373. doi: 10.1016/S0168-583X(97)00228-0
Mesjasz-Przybylowicz J, Przybylowicz WJ, Rama DBK, Pineda CA
(1997c) Elemental distribution in the Ni hyperaccumulator —
Senecio anomalochrous. Proceedings of the Microscopy Society of
South Africa 27, 89.
Mesjasz-Przybylowicz J, Przybylowicz WJ, Pineda CA (1998)
Elemental distribution in young leaves of the Ni hyperaccumulator
— Berkheya coddii. Proceedings of the Microscopy Society of
South Africa 28, 57.
Mesjasz-Przybylowicz J, Przybylowicz WJ, Rama DBK, Pineda CA
(2001a) Elemental distribution in Senecio anomalochrous, a Ni
hyperaccumulator from South Africa. South African Journal of
Science 97, 593–595.
Mesjasz-Przybylowicz J, Przybylowicz WJ, Pineda CA (2001b) Nuclear
microprobe studies of elemental distribution in apical leaves of the
Ni hyperaccumulator Berkheya coddii. South African Journal of
Science 97, 591–593.
Mizuno N, Nosaka S, Mizuno T, Horie K, Obata H (2003) Distribution
of Ni and Zn in the leaves of Thlaspi japonicum growing on
ultramafic soil. Soil Science and Plant Nutrition 49, 93–97.
Murray CG (1969) The petrology of the ultramafic rocks of
the Rockhampton district, Queensland. Geological Survey of
Queensland Publication No. 343.
Noell I, Morris D (1996) Localisation of hyperaccumulated nickel
in Stackhousia tryonii using electron-probe microanalysis.
In ‘Proceedings of microscopy and microanalysis’. (Eds
GW Bailey, JM Corbett, RVW Dimlich, JR Michael, NJ Zaluzec)
pp. 92–93. (San Francisco Press Inc: San Francisco)
Noell I, Morris D (1997) Nickel concentrations and plant defence in
Stackhousia tryonii. In ‘The ecology of ultramafic and metalliferous
areas’. Proceedings of the second international conference on
serpentine ecology. (Eds T Jaffré, RD Reeves, T Becquer)
pp. 109–110. (ORSTOM: Nouméa, New Caledonia)
Przybylowicz WJ, Pineda CA, Prozesky VM, Mesjasz-Przybylowicz J
(1995) Investigation of Ni hyperaccumulation by true elemental
imaging. Nuclear Instruments and Methods in Physics Research.
Section B, Beam Interactions with Materials and Atoms 104,
176–181. doi: 10.1016/0168-583X(95)00445-9
Przybylowicz WJ, Mesjasz-Przybylowicz J, Pineda CA, Churms CL,
Springhorn KA, Prozesky VM (1999) Biological applications of
the NAC nuclear microprobe. X-Ray Spectrometry 28, 237–243.
1074
Functional Plant Biology
N. P. Bhatia et al.
Przybylowicz WJ, Mesjasz-Przybylowicz J, Pineda CA, Churms
CL, Ryan CG, Prozesky VM, Frei R, Slabbert JP, Padayachee J,
Reimold WU (2001) Elemental mapping using proton-induced
X-rays. X-Ray Spectrometry 30, 156–163.
Psaras GK, Constantinidis T, Cotsopoulos B, Manetas Y (2000)
Relative abundance of nickel in the leaf epidermis of eight
hyperaccumulators: evidence that the metal is excluded from
both guard cells and trichomes. Annals of Botany 86, 73–78.
doi: 10.1006/ANBO.2000.1161
Reeves RD (1992) Hyperaccumulation of nickel by serpentine
plants. In ‘The vegetation of ultramafic (serpentine) soils’.
Proceedings of the first international conference on serpentine
ecology. (Eds AJM Baker, J Proctor, RD Reeves) pp. 253–277.
(Intercept Ltd: Andover)
Reeves RD, Baker AJM, Borhidi A, Berazain R (1996) Nickel
accumulating plants from the ancient serpentine soils of Cuba. New
Phytologist 133, 217–224.
Reeves RD, Baker AJM, Borhidi A, Berazain R (1999) Nickel
hyperaccumulation in the serpentine flora of Cuba. Annals of
Botany 83, 29–38. doi: 10.1006/ANBO.1998.0786
Robinson BH, Lombi E, Zhao FJ, McGrath SP (2003) Uptake and
distribution of nickel and other metals in the hyperaccumulator
Berkheya coddii. New Phytologist 158, 279–285.
Ryan CG (2001) Developments in dynamic analysis for quantitative
PIXE true elemental imaging. Nuclear Instruments and Methods
in Physics Research. Section B, Beam Interactions with Materials
and Atoms 181, 170–179. doi: 10.1016/S0168-583X(01)
00374-3
Schoonhoven LM, Jermy T, van Loon JJA (1998) ‘Insect-plant biology:
from physiology to evolution’. (Chapman and Hall: London)
Severne BC (1974) Nickel accumulation by Hybanthus floribundus.
Nature 248, 807–808.
Vázquez MD, Barceló J, Poschenrieder C, Madico J, Hatton P, Baker
AJM, Cope GH (1992) Localization of zinc and cadmium in
Thlaspi caerulescens (Brassicaceae), a metallophyte that can
hyperaccumulate both metals. Journal of Plant Physiology 140,
350–355.
Vázquez MD, Poschenrieder C, Barceló J, Baker AJM, Hatton P, Cope
GH (1994) Compartmentation of zinc in roots and leaves of the
zinc hyperaccumulator Thlaspi caerulescens J & C Presl. Botanica
Acta 107, 243–250.
Vergnano Gambi O (1967) Primi dati sulla localizzazione istologica
del nichel in Alyssum bertolonii desv. Giornale Botanico Italiano
101, 59–60.
Witkowski ETF, Weiersbye-Witkowski IM, Przybylowicz WJ,
Mesjasz-Przybylowicz J (1997) Nuclear microprobe studies of
elemental distributions in dormant seeds of Burkea africana.
Nuclear Instruments and Methods in Physics Research.
Section B, Beam Interactions with Materials and Atoms 130,
381–387. doi: 10.1016/S0168-583X(97)00231-0
Zhao FJ, Lombi E, Breedon T, McGrath SP (2000) Zinc
hyperaccumulation and cellular distribution in Arabidopsis
halleri. Plant, Cell and Environment 23, 507–514. doi: 10.1046/
J.1365-3040.2000.00569.X
Zierold K, Hagler HK (1989) ‘Electron probe microanalysis applications
in biology and medicine.’ (Springer Verlag: Berlin)
Manuscript received 22 October 2003, received in revised form 5 April
2004, accepted 21 June 2004
http://www.publish.csiro.au/journals/fpb