Plant Soil (2009) 314:35–48
DOI 10.1007/s11104-008-9703-4
REGULAR ARTICLE
Interaction of nickel and manganese in accumulation
and localization in leaves of the Ni hyperaccumulators
Alyssum murale and Alyssum corsicum
C. Leigh Broadhurst & Ryan V. Tappero &
Timothy K. Maugel & Eric F. Erbe &
Donald L. Sparks & Rufus L. Chaney
Received: 15 January 2008 / Accepted: 24 June 2008 / Published online: 2 August 2008
# US Government 2008
Abstract The genus Alyssum contains >50 Ni hyperaccumulator species; many can achieve >2.5% Ni in
dry leaf. In soils with normal Mn levels, Alyssum
trichome bases were previously observed to accumulate
Ni and Mn to high levels. Here we report concentration
and localization patterns in A. murale and A. corsicum
grown in soils with nonphytotoxic factorial additions of
Ni and Mn salts. Four leaf type subsets based on size
and age accumulated Ni and Mn similarly. The greatest
Responsible Editor: Fangjie J. Zhao.
Use of trade or proprietary names or trademarks herein is for
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advertisement or endorsement by the US Department of
Agriculture, University of Maryland, or University of
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Electronic supplementary material The online version of this
article (doi:10.1007/s11104-008-9703-4) contains
supplementary material, which is available to authorized users.
C. Leigh Broadhurst (*) : R. L. Chaney
US Department of Agriculture Henry A. Wallace
Agricultural Research Center, Environmental Management
and Byproduct Utilization Laboratory, Animal and Natural
Resources Institute,
Bldg. 007,
Beltsville, MD 20705, USA
e-mail: leigh.broadhurst@ars.usda.gov
R. V. Tappero : D. L. Sparks
Plant and Soil Sciences Department,
University of Delaware,
Newark, DE, USA
Mn accumulation (10 times control) was observed in A.
corsicum with 40 mmol Mn kg−1 and 40 mmol Ni kg−1
added to potting soil. Whole leaf Ni concentrations
decreased as Mn increased. Synchrotron X-ray fluorescence mapping of whole fresh leaves showed localized
in distinct high-concentration Mn spots associated with
trichomes, Ni and Mn distributions were strongly
spatially correlated. Standard X-ray fluorescence point
analysis/mapping of cryofractured and freeze-dried
samples found that Ni and Mn were co-located and
strongly concentrated only in trichome bases and in
cells adjacent to trichomes. Nickel concentration was
also strongly spatially correlated with sulfur. Results
indicate that maximum Ni phytoextraction by Alyssum
may be reduced in soils with higher phytoavailable Mn,
and suggest that Ni hyperaccumulation in Alyssum
species may have developed from a Mn handling
system.
T. K. Maugel
Laboratory for Biological Ultrastructure,
Department of Biology, University of Maryland,
College Park, USA
E. F. Erbe
Electron Microscopy Unit, Soybean Genomics
and Improvement Laboratory, Plant Sciences Institute,
USDA Beltsville,
Beltsville, MD, USA
36
Keywords Alyssum . Hyperaccumulator .
Manganese localization . Nickel localization .
Phytoremediation . trichomes
Introduction
More than 400 plant species are known to naturally
accumulate high levels of metals such as Cd, Cu, Co,
Mn, Ni and Zn. Hyperaccumulation is defined as
accumulation of >1000 μg g−1 in plant dry material
for Ni, and Co, and >10,000 μg g−1 for essential trace
elements such as Mn and Zn which are required in
larger amounts to support normal metabolism (Baker et
al. 2000). The genus Alyssum (Brassicaceae) contains
more than 50 Ni hyperaccumulator species, many of
which can achieve 3% dry weight Ni in leaf biomass
(Reeves 1992). Our research consortium has developed
commercially feasible phytoremediation and phytomining technologies that can potentially clean up Nicontaminated soils (Chaney et al. 1999, 2007; Li et al.
2003a, b). The technology employs the Ni-hyperaccumulating species Alyssum murale and A. corsicum
to phytoextract Ni from a range of soil types.
These species are endemic to serpentine (ultramaficderived) soils throughout Mediterranean Europe, but
unlike many serpentine-endemic species, they grow
rapidly and prolifically, even in nonnative environments
(Li et al. 2003a, b; Broadhurst et al. 2004a; Bani et al.
2007). Further, A. murale and A. corsicum can hyperaccumulate Ni in nonserpentine soil types, such as
limestone and organic soils (Küpper et al. 2001; Li et
al. 2003a, b; Broadhurst et al. 2004a). Several other
hyperaccumulator species such as Berkheya coddi
(Robinson et al. 2003) and A. serpyllifolium (De La
Fuente et al. 2007) have potential for phytoremediation/phytomining but have not yet been adequately
field tested. In a field test where climatic conditions
permitted growth of both Alyssum and Berkheya,
Berkheya accumulated <50% of the Ni concentration
that Alyssum achieved (Chaney et al. 2007).
Ni localization patterns have been determined for 12
Alyssum Ni hyperaccumulator species/ecotypes
(Krämer et al. 1997; Küpper et al. 2001; Psaras et al.
2000; Kerkeb and Krämer 2003; Marmiroli et al. 2003;
Broadhurst et al. 2004a, b; McNear et al. 2005;
Asemaneh et al. 2006; De La Fuente et al. 2007; Smart
et al. 2007; Tappero et al. 2007). Nickel is mainly
stored in the leaves, and is particularly concentrated in
Plant Soil (2009) 314:35–48
epidermal cell vacuoles, trichome bases, and the lower
parts of the trichome pedicle. The upper and lower leaf
surfaces of Alyssum sp. are covered with an overlapping
network of branching, stalked bifurcate or stellate
trichomes (Inamdar 1983; Oran 1996; Broadhurst et
al. 2004b). Trichomes are anchored in the epidermal
layer with a ~20 μm smooth cylindrical pedicle which
has a broad fan-shaped basal compartment.
In A. murale, the trichome is unicellular. The
trichome basal compartment and the epidermal cells
adjacent to the trichome basal compartment strongly
concentrate Ni, but only one (Smart et al. 2007) of
twelve studies has reported appreciable Ni in the
trichome rays or nodules. In soils with normal Mn
levels, Ni and Mn can be simultaneously accumulated
to very high levels within Alyssum trichome bases
(Broadhurst et al. 2004a, b; McNear et al. 2005;
Tappero et al. 2007) although the average leaf Mn
concentration is below the accepted threshold defining
hyperaccumulation. On the Alyssum leaf surface,
Tappero et al. (2007) found Mn concentrated in a
ring-like pattern surrounding the trichome base. To further quantify potential interactions between Ni and Mn,
we have investigated A. murale and A. corsicum grown
in soils with factorial additions of Ni and Mn salts.
Materials and methods
Horticulture
Alyssum murale ‘Kotodesh’ (from Albania) and
Alyssum corsicum (from Turkey) were started from
seed 14 Oct. 2005 in a greenhouse at USDA Beltsville. Forty-four days later seedlings were transplanted to prepared and equilibrated 500 ml pots
containing Promix® soil with an increasing series of
NiSO4·6H2O (0, 10, 40 mmol Ni kg−1) and/or
MnSO4·H2O additions (0, 10, 40 mmol Mn kg−1)
such that every combination was represented (c.f.
Table 1). Carbonates (half CaCO3 and half MgCO3)
were added to each pot at amounts equimolar with
NiSO4 + MnSO4 to buffer the acidity generated by
the metal salt additions. Initial soil pH was uniformly
6.4. Duplicate controls were prepared to better
establish background Mn accumulation in Alyssum.
The experiments were conducted in a greenhouse
under controlled temperature and light conditions and
ambient humidity. Photoperiod was 15:9 h day/night.
Plant Soil (2009) 314:35–48
37
Table 1 Nickel and manganese concentrations in Alyssum murale ‘Kotodesh’ and Alyssum corsicum leaves, mg kg−1 dry weight from
a composite of apical and distal leaves sampled in early spring
Treatment
Ni0/Mn0a
Ni0/Mn0a
Ni0/Mn10
Ni0/Mn40
Ni10/Mn0
Ni10/Mn10
Ni10/Mn40
Ni40/Mn0
Ni40/Mn10
Ni40/Mn40
A. murale
A. corsicum
Ni
Mn
Ni
Mn
30
45
92
32
10,100b
6,347b
5,930b
15,100b
12,300b
8,638
149
159
597
383
277
382
322
269
439
767
21
16
36
61
8,770b
9,330b
5,380b
15,300b
13,900b
12,600
96
80
334
306
187
285
602
257
695
924
Treatment Ni/Mn designates mmolkg−1 Ni and/or Mn salts added to Promix growth medium
a
Replicate control plants
b
Indicates Ni concentrations significantly higher than means for the same plant in Table 2
During this time supplemental high-intensity sodium
and incandescent lights capable of supplying
400 μmol m−2 s−1 supplemented sunlight if necessary.
Daytime temperature was 24°C with cooling initiated
at 27°C. Nighttime temperature was 18°C with cooling initiated at 21°C. Plants were grown in freely
drained plastic pots with saucers to prevent loss of
leachate, and watered with deionized water. The
plants received standard fertilization (100 mg N,
P2O5, K2O) once every two weeks.
All plants grew normally with no signs of
phytotoxicity. There were no significant differences
in biomass, rate of growth, growth habit or leaf size
among the various treatments. On 26 May (A. murale)
and 12 June 2006 (A. corsicum) they were transplanted to 1,000 ml pots with the same Ni/Mn levels
and grown until examined.
Leaf harvest
Two cm lengths of apical stem tip and leaves from
various locations on the stem were harvested on 23 Mar.
2006 for initial Ni and Mn analysis. Leaves for cryogenic
electron microscopy and standard energy dispersive Xray analysis (SEM/EDX) were harvested simultaneously.
A second harvesting was done 27 July 2006 to
establish whether leaf age (and related size) impacted Ni
and Mn concentrations. This harvesting was done
within a week of high-energy X-ray analysis. For each
plant, four leaf subsets were sampled as follows: (1)
youngest, smallest leaves from apical rosette; (2) young
relatively small singular leaves from just below the
apical rosette; (3) intermediate size and age leaves,
representing the mode of leaves on the plant; (4) oldest
largest leaves from stem areas distal to the apical rosette.
Multiple leaves of each subset were collected from each
plant at one sampling session. Because sampling took
place in July, some of the plants were stressed.
All the Alyssum hyperaccumulators we have grown
are vigorous, fast-growing and disease-resistant;
however, because they are native to Mediterranean
climatic zones they become stressed under high heat/
humidity conditions. All plants we have grown thus
far do relatively poorly in the Beltsville, MD
greenhouse during July, August, and September,
becoming prone to fungal infections and insect
infestations, and dropping leaves (plants grown
outdoors with good drainage and full sun are not
affected). Symptoms are exacerbated in plants not
allowed to hyperaccumulate Ni.
Total metals analysis
Harvested leaves were rinsed in deionized water to
remove any adhering soil particles. Leaf samples for
each treatment were dried for 24 h at 60°C, weighed,
and ashed in a 480°C oven for 16 h. After cooling, the
ash was digested with 2 ml concentrated HNO3 and
heated to dryness. The sample was then dissolved in
10 ml 3 N HCl, filtered through Whatman #40 filter
38
paper and brought to volume in a 25 ml volumetric
flask using 0.1 N HCl. For quality control, reagent
blanks and an in-house Alyssum standard were included. Ni and Mn were determined by inductivelycoupled plasma atomic emission spectrometry using
40 mg L−1 yttrium as an internal standard in all
samples and standard solutions.
Scanning electron and standard X-Ray fluorescence
spectrometry (SEM/EDX)
For microanalysis bulk A. corsicum leaf samples of
intermediate size (#3 as described above) were frozen
in liquid nitrogen and fractured to yield complement
cross-sections. A. corsicum has the highest trichome
density of 12 Alyssum hyperaccumulator species that
have been studied in detail, so we utilized it to
maximize our chances of sectioning trichome bases.
The trichomes are distributed equally over upper and
lower epidermii (Fig. 1 a); however the physiology of
the trichomes and adjacent cells differs between upper
vs. lower epidermis. On the upper epidermis trichomes ascend from cup-like depressions formed by
the adjacent cells. The cup-shaped feature is much
less distinct on the lower epidermis but can still be
recognized (Fig. S1; see supplemental information).
The cupped structure allowed us to identify trichome
bases easily (especially on the upper epidermis) even
when the trichome itself had broken off.
One half of each complement remained frozenhydrated and was etched and imaged with SEM at
2.0 kV. The other half was freeze-dried at −80°C
(Pearse–Edwards tissue drier) and examined with
SEM/EDX at 20 kV. Details of our procedure are
given in Broadhurst et al. (2004a).
For EDX analysis, we utilized only the four treatments with both Ni and Mn additions. Freeze-dried
complement fracture leaf sections were individually
mounted on standard aluminum stubs coated with silver
adhesive paste. Samples were placed on edge to view
the cross sections that correspond to the complement
fracture planes. Before analysis a light coat of carbon
was deposited on the samples at a pressure of 13 Pa
followed by 20 nm of aluminum deposited at 1.3 10−3
Pa. In order to control excessive charging, samples
were first analyzed as is and then some trichomes
gently scraped off with a glass rod. Data in Table 2
include analyses both with and without trichomes,
however the maps presented in Figs. 4 and S4. are
Plant Soil (2009) 314:35–48
Fig. 1 a Alyssum corsicum cryogenic complement fracture
showing cross section and lower epidermis. b Whole fresh leaf
SXRF map for Ni40/Mn40 plant. Left panel shows Ni, Mn, Ca
maps and the tricolor map. Right panel shows the correlation
for Ni Kα vs. Mn Kα counts for a selected trichome (masked
on left) and the correlation for the whole leaf area (inset graph).
Due to the extensive network of trichomes, Ca signal covers
nearly 100% of the image, so for imaging purposes a very high
threshold of Ca signal intensity was set
exclusively from samples with trichomes removed.
Mapping was rendered imprecise without removing
some trichomes due to motion of the sample over the
long duration in the electron beam.
We used an Amray 1820D scanning electron
microscope (KLA Tencor Corp., AMRAY Division,
Bedford, MA), an EDAX ECON 4 detector with an
active area of 10 mm and an EDAX DX Prime
analyzer (EDAX Inc, Mahwah, NJ). The samples
were analyzed under the following conditions: 15–
20 kV accelerating voltage, 20° tilt, 12 mm working
distance, 17.66° takeoff angle. Each spot location was
counted for 100 live seconds. Beam spot was 1 to
10 μm depending on the application, but always less
than a cell diameter when targeting a specific cell/
location. Semiquantitative results were corrected
using ZAF algorithms. Every location that was
analyzed was also digitally photographed with the
AMRAY instrument.
Elemental X-ray maps of Ni, Mn, Ca, Cl, K, Mg, P,
S and Si signal were done for a few locations that
afforded excellent cross sections of individual trichome bases. X-ray compositional analysis and
mapping were accomplished with a windowless
detector using EDAX Genesis software. Elemental
mapping was done at a dwell time of 200 μs, a time
constant of 50 μs, and resolutions of either 256 × 200
or 512 × 400 pixels.
Synchrotron X-ray fluorescence spectrometry (SXRF)
SXRF images of whole fresh leaves were acquired at
beamline X-27A of the National Synchrotron Light
Source at Brookhaven National Laboratory, Upton,
NY. This beamline uses Kirkpatrick–Baez mirrors to
produce a focused 10–15 μm spot of hard X-rays with
tunable energy achieved via a Si(111) monochromator. For SXRF imaging, the incident energy was fixed
at 10 keV to excite all target elements simultaneously.
Live plants were transported to the synchrotron
facility in their pots, and fresh leaves of size #2 were
b
Plant Soil (2009) 314:35–48
excised from plants immediately prior to analysis.
Leaves were mounted onto Kapton® tape and
attached to the sample stage with no further
preparation. Specimens were rastered in the path of
39
the beam by an XY stage oriented 45° to the
incident beam, and X-ray fluorescence was detected
by a 13-element Ge detector (Canberra) positioned
90° to the beam.
40
Plant Soil (2009) 314:35–48
Table 2 Nickel and manganese concentrations in Alyssum murale ‘Kotodesh’ and Alyssum corsicum whole leaves, mg kg−1 dry
weight from ICP−AES, and interaction between Mn and Ni
Treatment
A. murale
A. corsicum
Ni (mmol/kg)
Mn (mmol/kg)
Foliar Mn (mg/kg)
Foliar Ni (mg/kg)
Foliar Mn (mg/kg)
Foliar Ni (mg/kg)
0
0
0
10
10
10
40
40
40
0
10
40
0
10
40
0
10
40
102e
408c
461c
198de
649b
710b
281d
424c
1,320a
22d
53d
48d
3,430c
3,640c
2,490c
11,200a
9,710ab
8,770b
82d
220bc
510a
103cd
318b
240b
112cd
566a
NSa
<10d
40d
<10d
2,760c
4,520b
1,070d
8,300a
8,320a
NS
Means followed by the same letter are not significantly different at P<0.05
According to the Waller–Duncan K-ratio t test
Entries are the mean metal concentration in four leaf type subsets from each plant sampled in the summer at time of SXRF analysis. Ni
foliar levels were significantly negatively affected by both Mn soil additions and hot, humid growing conditions. Less than ten
indicates concentration below detection limits.
a
Ni40/Mn40 plant remained at the University of Delaware so was unavailable for sampling.
Elemental maps were typically collected from a 1 to
3 mm2 area using a step size of 20 μm and a dwell time
of 500 ms. Smaller areas such as individual trichomes
were imaged at ∼10 μm. Leaf areas larger than 3 mm2
were not imaged because of the time required.
Manganese and Ni X-ray absorption near-edge structure
(μ-XANES) spectra were collected at points of interest
selected from the SXRF images, as detailed in Tappero
et al. (2006). The monochromator was calibrated to the
Mn (6539 eV) or Ni (8333 eV) K-edge energy with
metallic foils, and spectra were collected from 200 eV
below the absorption edge to k values of 9 A−1.
Results
Ni and Mn concentrations
Nickel and Mn concentrations for the composite
spring leaf sample are given in Table 1. Both A.
murale and A. corsicum hyperaccumulated Ni and
accumulated high levels of Mn when higher exogenous levels of these metals were provided; both
species followed the same accumulation patterns.
The mean values of the leaf subsets (Table 2) follow
the same trend as the composite samples but the
maximum Ni accumulation was significantly lower.
Mn levels in the leaves uniformly increased over
background as soil Mn levels increased. The back-
ground Mn levels in the control A. murale samples
were significantly higher than the corresponding A.
corsicum controls; both controls were Promix hence
Mn-sufficient. The highest Mn accumulation
(924 mg kg−1), observed in A. corsicum Ni40/Mn40,
was approximately ten times higher than control.
For the Ni40 plants, leaf Ni concentrations decreased as Mn concentrations increased. At Mn40
levels neither species was able to accumulate as much
Ni as Mn0 plants did (Tables 1, 2). Interestingly, for
both species, the highest Mn concentrations were
observed in the Ni40/Mn40 plants, not the Ni0/Mn40
plants—the reverse of the situation with Ni. For the
Ni10 plants, Ni generally decreased as Mn increased
with the exception that the Ni10/Mn10 plants had
higher Ni than the Ni10/Mn0 plants.
For the four leaf type subsets, there was no
significant pattern in Ni and Mn accumulation with
respect to size, age, or position on the stem. Our data
indicate that leaves begin to hyperaccumulate Ni and
accumulate Mn as soon as they emerge and continue
to do so as they grow and age. Therefore both leaf
sizes (#2, #3) that were X-ray analyzed can be
considered representative of the plant as a whole.
Overall, the composite samples accumulated nearly
twice as much Ni as the leaf type subset mean. This
almost certainly reflects sampling in March vs. late
July, when the plants were stressed, as discussed in
Materials and Methods.
Plant Soil (2009) 314:35–48
There may be a tendency for leaf type 3 to contain the
greatest amount of Ni but clearly identifying this pattern
would require growing and analyzing multiple healthy,
mature plants at each Ni/Mn addition level, which is
beyond the present scope. Type 3 represents the mode of
leaves on healthy plants, however Type 3 leaves may
not be the most abundant on stressed plants. Stressed
plants tend to drop lower leaves, sometimes retaining
only the apical rosette. This is especially true for A.
corsicum, which has relatively large, thick leaves and
stems—hence many fewer leaves and stems occur on a
given plant as compared to A. murale, A. pterocarpum,
A. pintodasilva and other species.
SXRF analysis
The epidermis of each whole fresh leaf we examined
showed the same basic pattern of Mn concentrations
localized in distinct spots that are associated with
trichome bases (Figs. 1b, S3). The stellate distributions of high Ca concentration are trichome rays,
which are filled or covered with Ca carbonate and/or
oxalate crystallites, and yield X-ray signals from Ca,
Mg, C and O only. The entire leaf surface of A.
corsicum is very densely packed with trichomes that
form a layer 20–30 μm above the leaf epidermis
(Figs. 1a, S1). The Ca X-ray fluorescence is so
intense from this layer on flat-leaf images that it
essentially masks underlying signals from other
elements. Therefore we have shown representative
Ca signal only from a few trichomes in Figs. 1 b and
S3 in order that the Ni and Mn data remain visible.
The typical intense Ca signal from the trichome rays
can be seen along the leaf margin in these figures.
Nickel and Mn distributions are spatially correlated
but not homogenously distributed. Figure 1b
(Ni40Mn40) shows somewhat reduced Ni signal
intensity as compared to Fig. S3, (Ni10Mn10), in
agreement with the whole leaf Ni data (Table 1).
Linear areas of higher Ni concentration near the
center of the leaf roughly correspond to epidermal
cells overlying vascular tissue; however with this
technique Ni signal is detectable across the entire leaf
surface.
Analysis of the XANES spectrum from a trichome
area clearly identified Mn2+ as the predominant Mn
oxidation state (Fig. 2). Similarly, the Ni XANES
spectrum of the general leaf epidermis shows that Ni
is present as Ni2+ (Fig. S2).
41
Semiquantitative SEM-EDX analysis
The SEM-EDX analysis focused on a few locations with
images no larger than 40 × 20 μm in order to better
understand the generalized Ni and Mn distribution
pattern described above. Further, the cryogenic complement fracture method cleanly shears cells, or removes
discs of cell membrane so that cell interiors can be
accessed and distinguished clearly from cell walls.
Larger scale imaging and analysis is needed to identify
the many locations where cellular/vacuolar contents are
wholly or partially absent. When the vacuole is
“plucked out” of an epidermal cell during sample
preparation or handling, the resultant concavity typically retains a residue which clings to the inner cell wall.
This residue can be moderately high in Ni (i.e.
significantly above EDAX detection limits), thereby
leading to the (incorrect) conclusion that Ni is sequestered in cell wall, yet this residual Ni concentration is far
lower than that of the intact vacuole. Table 3 and Figs. 3
and S4 provide examples of this situation.
We focused primarily on cleanly sectioned trichomes
and the epidermal cells immediately adjacent to
trichomes. Ni was generally concentrated in upper and
lower epidermal cells (Table 3). Please note that data in
Table 3 are semiquantitative only. Approximate weight
percents are given solely to facilitate understanding of
Ni and Mn localization patterns and should not be
construed as defining hyperaccumulation levels. The
achievement of hyperaccumulation is based on the
ICP-AES data for dry leaves (Tables 1 and 2).
Nickel and Mn were both strongly concentrated in
trichome bases and lower pedicles, and in cells adjacent
to the trichomes (Figs. 3 and 4). As observed in the
whole leaf, Ni and Mn were co-located in the trichome
bases and adjacent cells but the maximum Ni
concentration observed in a location did not always
correspond with the maximum Mn concentration. In
particular, the upper parts of trichome bases tended to
have high Ni without much Mn accumulation..
Figure 3 confirms in cross-section the ring-like
distribution of Mn observed by Tappero et al. (2007).
In general, trichomes on the epidermis which overlay
the main vascular bundle were observed to have
vacuoles strongly enriched in Ni. Mn was at or below
detection limits in cells below the epidermal layer, in
trichome rays, and in general epidermal cells that
were not associated with a trichome. Manganese must
be present in all chloroplasts, but the typical foliar
42
Plant Soil (2009) 314:35–48
Fig. 2 Top left panel shows tricolor Ni–Mn–Ca elemental map
for whole fresh leaf from a Ni40/Mn40 plant. The inset box
delineates transect (1) across a trichome near the leaf margin.
The line spectra across the transect verifies the ring-like
distribution of Mn and Ni in trichome bases. On the top right
panel, the μ-XANES spectra of spot 1 are compared to Mn II
and Mn IV standards, demonstrating that the spot 1 absorption
edge is similar to the Mn II standard
concentrations are low compared to the levels in the
trichome base spots.
Sulfur was strongly correlated with Ni in the Ni40
samples (Fig. 4). For each individual line spectrum
we collected, high Ni signal was always associated
with much greater than average S signal intensity. We
found no additional correlations between Ni or Mn
and K, Cl, P or Si. The central area of Fig. S4 has a
concavity which corresponds to the projection of the
tip of trichome base in the complement fracture. Some
material from the trichome base has been retained,
and in a smaller scale image would probably be
identified as mesophyll. The Ni and S are signals are
strongest in the intact upper epidermis, but Ni and S
also appear somewhat dispersed. The Ni Lα signal is
particularly strong since that area is not adjacent to
Plant Soil (2009) 314:35–48
43
Table 3 Representative peak/background count ratios expressed as ZAF-corrected semiquantitative weight percents for Ni and Mn in
trichome bases and adjacent cells from EDX point analyses (A. corsicum)
Sample/Location
Ni10/Mn10
ADJ C (3)
TBa basal
TBa mid
TBa apical
TBb apical
Ni40/Mn10
ADJ C (3)
TB (5)
TB cell wall
Ni10/Mn40
ADJ C (7)
TBa basal
TBa mid
TBa apical
TBb basal
TBb mid
TBb apical
Ni40/Mn40
ADJ C (6)
epidermal cella
Mesophylla
Mesophyllb
TB (5)
TB basal
TB mid 1
TB mid 2
TB apical
Ni wt%
Mn wt%
Comment
9.0±1
8.0
6.1
6.6
4.7
DL
DL
DL
2.0
2.1
high concentration area (HCA)
HCA
9.0±1
8.9±2
1.9
DL
DL
DL
94% C and O; low metals
4.5±2
3.3
DL
DL
9.0
DL
DL
2.5±1
8.7
DL
DL
12
1.4
DL
11±4
27
4.6
2.4
4.2±2
2.3
4.5
2.3
19
8±5
1.2
DL
DL
1.9±0.5
DL
1.4
1.9
1.9
HCA
HCA
2 cells away from TB listed below
spongy, next to ADJ
spongy, wide area scan
whole TB scans
HCA but contents mainly absent
HCA but contents mainly absent
above mid 1,” “
HCA, contents present
In general mesophyll and vascular tissue yielded insignificant Ni and Mn counts so data are not reported. Values in parentheses
indicate number of locations averaged. ADJ C -adjacent cell; TB-trichome base. When possible trichome bases were analyzed
stepwise from the basal area upwards.
DL values at or below 1%
a
General epidermal cells not directly associated with trichomes always had insignificant Mn counts and may or may not have
significant Ni counts, depending on the Ni soil level. This cell had a very large intact vacuole. It may have been an adjacent cell for
another trichome but it could not be determined.
the trichomes, which can cause scattering of lower
energy X-rays. We also observed that on occasion
both Ca and Mg were strongly concentrated in the
trichome rays in a ratio consistent with dolomite.
Discussion
Nickel and Mn concentration and distribution patterns
in A. corsicum resulting from the factorial additions of
Ni and Mn salts confirm previous observations of
Alyssum species in non-Mn enriched soils. Previously
we found that Mn and Zn levels were elevated in
Alyssum leaves across a series of Ni additions (10–
90 mmol Ni kg−1) as compared to control and Nianalytical standard plants (Broadhurst et al. 2004a).
Concentrations in duplicates of A. murale grown in
Promix without Mn addition were as follows: for Ni10
plants: 10,900 and 9,470 mg Ni kg−1; 286 and
389 mg Mn kg−1. For Ni40 plants: 17,300 and
17,000 mg Ni kg−1; 279 and 223 mg Mn kg−1. Data
for Mn0 treatments in this study were comparable.
We first detected localized high concentrations of
Mn in A. murale ‘Kotodesh’ plants grown in natural
serpentine soil (Kukier and Chaney 2001; Broadhurst
et al. 2004b). Leaves from these plants contained 8040
44
Plant Soil (2009) 314:35–48
spot
Ni %
Mn %
1
3
nd
2
7
2
3
16
9
4
23
2
5a
23
20
5b
29
22
5c
27
20
6
17
18
7
47
2
8
8
nd
Fig. 3 a Trichome located on the lower epidermis in the
vicinity of the main vascular bundle. b Close up of the trichome
and adjacent cells. Semiquantitative oxygen-free Ni and Mn
weight percentages for the points designated are listed.
Unavoidable concave sample geometry resulted in low oxygen
counts but did not affect metal signals. Note that locations 1
and 2 have the bulk of the cellular contents missing; point 4 is
more representative of the trichome base Ni content overall. (nd
not detected)
mg Ni kg−1and 801 mg Mn kg−1. A. corsicum leaves
from plants grown in the serpentine soil contained
7600 Ni mg Ni kg−1 and 289 mg Mn kg−1. Similarly,
in the current study Mn levels in the control A. murale
plants were significantly greater than the corresponding
A. corsicum controls. However, both species concentrated Mn when it was provided at higher phytoavailable levels. Both Ni hyperaccumulation and Mn
accumulation are initiated when leaves are young and
continue at approximately the same rate as the leaf
ages.
The most important difference observed upon
addition of high soil Mn was that Ni foliar concentrations decreased as Mn foliar concentrations increased, indicating that increasing soil Mn levels
interfere with Ni uptake, translocation and/or storage.
Further, the greatest Mn concentrations were observed
in the Ni40/Mn40 plants, not the Ni0/Mn40 plants,
indicating that Ni did not interfere with Mn accumulation and may in fact potentiate it. Therefore
maximum Ni phytoextraction by Alyssum may be
reduced in soils with high phytoavailable Mn.
Low S may also decrease Ni phytoextraction. The
positive correlation between Ni and S localization was
strong, probably reflecting NiSO4 storage in epidermal vacuoles as suggested previously (Küpper et al.
2001; Broadhurst et al. 2004b). A similar correlation
of Ni and S X-ray counts and localization patterns
was observed in plants grown from hyperaccumulator
populations of A. bracteatum and A. murale but not in
control plants grown from a nonserpentine A. murale
population (Asemaneh et al. 2006). The Ni2+ oxidation state for hyperaccumulated Ni in the leaf
epidermis is consistent with NiSO4 storage but does
not rule out other ligands.
The strongly heterogenous Mn distribution pattern is
characteristic of many plants, which exhibit high Mn
“spots” on leaves when grown with high Mn supplied, as
opposed to uniform tissue Mn elevation (Blamey et al.
1986; Marschner 1995; Iwasaki and Matsumura 1999).
Manganese spots are generally described as a response
to Mn toxicity, since they eventually become necrotic
as Mn levels increase. Our Alyssum plants did not
exhibit symptoms of Mn phytotoxicity yet maintained
very tight control on the “spot” distribution pattern,
with high Mn concentrations restricted to trichome
bases and the cells immediately adjacent to trichomes.
We observed very high localized Ni and Mn
concentrations—sometimes over 20 dry wt%—only
Plant Soil (2009) 314:35–48
45
Fig. 4 SEM guide photo of A. corsicum Ni40/Mn40 plant (a).
Leaf cross section, lower epidermis; trichome base (trichome
broken off) and adjacent cells in center of image. X-ray maps
(20 KeV; Kα, β) show Ni (b), Mn (c) and S (d) distribution.
Map overlay software showed nearly 100% correlation between
Ni and S distribution
within trichome bases and intact vacuoles inside
epidermal cells associated with trichomes. As discussed previously, analyses must be undertaken
carefully to ensure that residues on the cell wall are
clearly distinguished from cell wall proper, since
these residues may still contain a respectable 5 wt%.
It is also imperative that quantitative whole leaf
analyses back up semiquantitive fluorescence mapping, so that total leaf Ni concentrations are known to
be >1,000 μg g−1. For example, the mean Ni dry wt%
for 13 epidermal spots analyzed by EDX reported in
Smart et al. (2007) is 0.29%, with 7 of the locations
containing undetectable concentrations. Since it is
clear that epidermal cells contain the bulk of the Ni in
Alyssum hyperaccumulator species, the corresponding
whole leaf concentrations cannot be within the realm
of hyperaccumulation, thus may bear little relevance
towards development of useful phytoremediation/phytomining technologies. Further, the standard ZAF
matrix corrections on these highly nonuniform biolog-
ical samples are not accurate enough that 0.29 wt% can
be considered a significant result. To err on the
conservative side, we do not report semiquantitative
weight percentages under 1% as detections.
The fresh whole leaf SXRF technique ensures that
metals are not redistributed during preparation for
analysis, so is an excellent check on other methods
which require sample dehydration and/or fixation. Leaf
folds and curls as observed herein and discussed in detail
in Tappero et al. (2007) are the major caveat. Also using
whole leaf SXRF and A. murale ‘Kotodesh,’ McNear et
al. (2005) reported high Ni concentrations associated
with vascular tissue which they explained as “entrainment during fixation as opposed to tissue loading.” We
observed a pattern consistent with this although we did
not have a whole leaf cross-section.
Previous authors are in agreement that Ni is not
stored in vascular tissue (Krämer et al. 1997; Küpper et
al. 2001; Psaras et al. 2000; Kerkeb and Krämer 2003;
Marmiroli et al. 2003; Broadhurst et al. 2004a, b;
46
Asemaneh et al. 2006; De La Fuente et al. 2007;
Tappero et al. 2007) yet the whole leaf fresh SXRF
data indicate a pattern of elevated Ni signal intensity
that roughly correlates with the vascular system. An
association of Ni with vascular tissue is logical given
that the vascular system must transport Ni and Mn. The
pattern of relatively higher Ni concentration associated
with the vascular system may be a combination of
preservation of Ni-rich fluids in transit as the leaf is
harvested, and an elevated concentration in storage
vacuoles in epidermal tissue/trichomes which overly
the vascular bundle.
Manganese concentration at the base of trichomes
has also been observed in sunflower (Blamey et al.
1986), pumpkin (Iwasaki and Matsumura 1999) and
cucumber (Horiguchi 1987). In these crop plants,
trichome Mn storage increased and Mn phytoxicity
decreased at a given Mn level when silicon was
supplied to the plants. Apparently, Si facilitates the
storage of Mn at the trichome base in a metabolically
inactive form. In the case of Alyssum, localized high
Mn concentrations are associated with trichomes at
much lower whole leaf Mn levels than present in the
other species—where Mn localization was observed
under Mn-phytotoxic conditions. Further, in whole
fresh Alyssum leaves Mn2+ is the predominant
oxidation state, not the insoluble, oxidized Mn4+.
These trichome-associated localization patterns
contrast with the pattern observed in the Mn hyperaccumulators/strong accumulators Gossia bidwillii,
Virotia neurophylla, Macadamia integrifolia and M.
tetraphylla, where the highest Mn concentration was
measured in palisade mesophyll (Fernando et al.
2006a,b). Further studies of the Ni–Mn interaction
in Ni-hyperaccumulators using Mn concentrations
approaching phytotoxicity to Alyssum will be necessary to understand the localization patterns.
Higher plants with two functionally distinct vacuoles
within a single cell have been reported by several
research groups (Paris et al. 1996; Jauh et al. 1999;
Epimashko et al. 2004; Epimashkoand Fischer-Schliebs
2006; Martinola et al. 2007). A model with two or more
discrete vacuoles within an epidermal cell fits the
observation that Ni and Mn were co-located in trichome
bases but accumulated in separate areas as opposed to
being interspersed. Our complete series of SEM photographs of frozen hydrated leaf cross sections does
contain images which have a visible demarcation line
across a trichome base or adjacent cell vacuole.
Plant Soil (2009) 314:35–48
The present findings raise the possibility that Ni
hyperaccumulation in Alyssum species originally developed from the Mn handling system. Most serpentine
soils contain high levels of Mn in the form of oxides.
The unexpected high Mn concentration in discrete
regions at the base of trichomes has no apparent
physiological basis, but it clearly occurs in Alyssum
Ni hyperaccumulators (Küpper et al. 2001; Broadhurst
et al. 2004b; McNear et al. 2005; Tappero et al. 2007).
This localized high-concentration Mn2+ storage suggests the presence of a tonoplast Mn permease specific
to trichome pedicles, while high epidermal cell vacuolar
Ni storage similarly suggests the presence of a tonoplast
Ni permease. Whether sulfate is also actively transported into these cells needs to be firmly established.
Also unexplained is the mechanism whereby trichome cells push Ca and Mg into the trichome rays
while storing Ni and Mn in the base. Yet Alyssum
murale is also selective enough that it excretes
(serpentine) geogenic Co exocellularly via hydathodes
(Tappero et al. 2007), and virtually excludes geogenic
Cr from uptake (Brooks 1987; Bani et al. 2007). De La
Fuente et al. (2007) also observed significant Mg along
with Ca in trichomes, suggesting that both these metals
are present in an inorganic form in trichome nodules. It
has been suggested that the serpentine-endemic Mnhyperaccumulators Alyxsia sp. and Austomyrtus bidwiilii accumulate Mn at the expense of Ca and Mg
(Brooks et al. 1981; Bidwell et al. 2002), so the
potential for interaction among all stored metals exists
for hyperaccumulator species in general.
These questions add to the remaining mysteries of
hyperaccumulators and require scientific explanation if
we are to obtain improved hyperaccumulator cultivars
for practical remediation of contaminated soils.
Acknowledgements We greatly appreciate the assistance of
USDA-ARS personnel Carrie Green for ICP analyses and
Christopher Pooley for computer graphics.
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