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REGULAR PAPER
Localization of aluminium in tea (Camellia sinensis) leavesusing low energy X-ray fluorescence spectro-microscopy
Roser Tolra • Katarina Vogel-Mikus • Roghieh Hajiboland • Peter Kump •
Paula Pongrac • Burkhard Kaulich • Alessandra Gianoncelli • Vladimir Babin •
Juan Barcelo • Marjana Regvar • Charlotte Poschenrieder
Received: 19 February 2010 / Accepted: 25 March 2010
� The Botanical Society of Japan and Springer 2010
Abstract Information on localization of Al in tea leaf
tissues is required in order to better understand Al tolerance
mechanism in this Al-accumulating plant species. Here, we
have used low-energy X-ray fluorescence spectro-micros-
copy (LEXRF) to study localization of Al and other low
Z-elements, namely C, O, Mg, Si and P, in fully developed
leaves of the tea plant [Camellia sinensis (L.) O. Kuntze].
Plants were grown from seeds for 3 months in a hydroponic
solution, and then exposed to 200 lM AlCl3 for 2 weeks.
Epidermal-mesophyll and xylem phloem regions of 20 lm
thick cryo-fixed freeze-dried tea-leaf cross-sections were
raster scanned with 1.7 and 2.2 keV excitation energies to
reach the Al–K and P–K absorption edges. Al was mainly
localized in the cell walls of the leaf epidermal cells, while
almost no Al signal was obtained from the leaf symplast.
The results suggest that the retention of Al in epidermal leaf
apoplast represent the main tolerance mechanism to Al in
tea plants. In addition LEXRF proved to be a powerful tool
for localization of Al in plant tissues, which can help in our
understanding of the processes of Al uptake, transport and
tolerance in plants.
Keywords Camellia sinensis (L.) O. Kuntze � Cell wall �Elemental spatial distribution � Epidermis � LEXRF �Synchrotron based X-ray fluorescence � Tea plant
Introduction
Aluminum (Al) is a ubiquitous element without a known,
specific, biological function. As one of the most abundant
elements in the Earth crust, after Si and O, it is present in
the daily life of all organisms (Berthon 2002; Poschenrie-
der et al. 2008). Al generally occurs as a component of a
variety of crystalline aluminosilicate-, oxyhydroxide- and
non-silicate-containing minerals, and it is thus usually
regarded as not being available for chemical and biological
reactions (Berthon 2002). However, under acidic condi-
tions (pH \ 4.5), Al is solubilised into a toxic trivalent
cation, Al3?, which is more available for biochemical
processes. Al3? has a high affinity for O2-donor com-
pounds, such as Pi, nucleotides, RNA, DNA, proteins,
carboxylic acids, phospholipids, polygalacturonic acid,
heteropolysaccharides, lipopolysaccharides, flavonoids and
antocyans, among others (Huang 1984; Martin 1986), and
even the very low concentrations of free Al3? in the
symplasm are potentially toxic (Ma et al. 1998). Indeed,
micromolar concentrations of Al3? can affect root growth
and development in many agriculturally important plant
species (Doncheva et al. 2005), and Al toxicity has been
recognized as a major factor that limits plant performance
R. Tolra � R. Hajiboland � J. Barcelo � C. Poschenrieder
Laboratorio de Fisiologıa Vegetal, Facultad de Biociencias,
Universidad Autonoma de Barcelona, 08193 Bellaterra, Spain
K. Vogel-Mikus (&) � P. Pongrac � M. Regvar
Department of Biology, Biotechnical Faculty,
University of Ljubljana, Vecna pot 111,
1000 Ljubljana, Slovenia
e-mail: katarina.vogel@bf.uni-lj.si
R. Hajiboland
Plant Science Department,
University of Tabriz, 51666 Tabriz, Iran
P. Kump � P. Pongrac
Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
B. Kaulich � A. Gianoncelli � V. Babin
Synchrotron Radiation Facility, Elettra–Sincrotrone Trieste,
S.S. 14, km163.5 in Area Science Park,
34149 Trieste-Basovizza, Italy
123
J Plant Res
DOI 10.1007/s10265-010-0344-3
in acidic soils (Kochian et al. 2004). Nevertheless, some
plants have adapted to this high Al3? availability in acidic
soils, especially in tropical regions, by developing resis-
tance or tolerance mechanisms (Jansen et al. 2002).
Among Al accumulators, the tea plant [Camellia sin-
ensis (L.) O. Kuntze, Theaceae] is the subject of intense
research. Studies of the localization and speciation of Al in
tea leaves aim to provide a better understanding not only of
Al-tolerance mechanisms in plants, but also of the fates and
risks of dietary Al intake in humans who consume tea-
based beverages (Carr et al. 2003; Morita et al. 2004). Al is
a neurotoxicant that can cause Fe-mediated oxidative stress
and cell injury (Yokel 2000) and although this is still under
debate, Al has also been linked to Alzheimer’s disease
(Exley 2007). Although the oral bioavailability of Al from
tea infusions has been estimated to be on average 0.37% in
rats, daily tea consumption can still provide a significant
amount of Al that reaches the systemic circulation (Yokel
and Florence 2008). There are, however, large differences
in Al content and Al leachability into infusions among the
varieties of teas produced (Fung et al. 2009), and increased
Al in tea leaves can increase the Al load in the diet (Carr
et al. 2003).
In tea plants, Al has been reported to be mainly accu-
mulated in old leaves, where up to 18-fold greater con-
centrations have been found compared to young leaves
(Carr et al. 2003). Al has been proposed to be translocated
from the roots to the shoots as an Al-citrate complex
(Morita et al. 2004), which also supports the hypothesis
that tea plants internally detoxify Al by forming non-toxic
Al complexes with organic acids and/or phenolic sub-
stances (Nagata et al. 1992; Ma 2000; Ma et al. 2001).
Complex formation of Al with organic acids suggests that
Al can be compartmentalised within leaf cell vacuoles, as
has been shown in localization studies for Ni, Zn and Cd in
some other hyperaccumulators (Ma et al. 2001; Vazquez
et al. 1992, 1994; Vogel-Mikus et al. 2008a, b). However,
pioneer microscopy studies on Al hyperaccumulation in tea
leaves already indicated a preferential accumulation in cell
walls of epidermal cells (Matsumoto et al. 1976). More
recently, investigations using energy-dispersive micro-
analysis (EDXMA) have also shown that in old tea leaves,
Al is mainly accumulated in the cell walls of epidermal
cells, while in younger tea leaves, the EDXMA was not
sufficiently sensitive to obtain Al localization maps or to
detect Al in symplast compartments (Carr et al. 2003).
Different analytical techniques have been developed to
date that are similar in terms of lateral resolution, chemical
sensitivity, quantitative analysis, depth profiling or bulk
sensitivity, and detection of elemental isotopes. Among
these techniques, the most widely used EDXMA in trans-
mission and scanning electron microscopy has provided the
greatest lateral resolution (\10 nm), although with a
moderate chemical sensitivity (0.01–0.1 wt%). In contrast,
although particle-induced X-ray emission (PIXE) can today
achieve lateral resolution in the micron range, it is not as
sensitive for the lower-Z elements and it can cause greater
beam damage to specimens than X-ray-induced methods
(Janssens et al. 1996). On the other hand, significant pro-
gress in the application of X-ray fluorescence (XRF)
analysis in biology was made through the development of
highly brilliant and energy-tunable synchrotron radiation
sources, and the possibility of focusing such X-ray light
down to sub-micron probes (Bertsch and Hunter 2001).
Laboratory and synchrotron-based XRF instrumentation
typically uses hard X-rays, and only a few soft X-ray XRF
set-ups have been reported (Flank et al. 2006). This is
related to the limitations to the use to date of soft X-rays
for XRF analysis, which include low fluorescence yield of
low-Z elements and unavailability of suited detectors and
electronics. The low-energy XRF system used in the
present study is operated at a soft X-ray regime (280–
2,200 eV). This is especially suited for bio-related
research, as it gives access to the elemental distribution of
the low-Z elements C, N, O, F, Al, Fe, Zn and Mg, and to
other elements that are of fundamental importance for
metabolism in biological systems at the cellular and/or sub-
cellular levels (Kaulich et al. 2009).
The aim of the present study was therefore to investigate
the spatial distribution of Al and other low-Z elements in
optimally developed leaves of tea plant using a novel tech-
nique—low-energy XRF (LEXRF) spectro-microscopy.
Materials and methods
Plant growth conditions
Tea [Camellia sinensis (L.) O. Kuntze] seeds collected in
autumn 2008 from tea gardens of Tea Research Institute,
Fashalem, North Iran, were germinated in moist vermicu-
lite at 25�C in dark. After emergence, the seedlings were
transplanted to continuously aerated low-ionic-strength
control nutrient solution (pH 4) with the following com-
position: 175 lM (NH4)2SO4, 325 lM NH4NO3, 23 lM
K2SO4, 124 lM CaCl2, 20 lM MgSO4, 12.5 lM KH2PO4,
82.5 lM MgCl2, 185 lM KCl, 11.5 lM H3BO3, 0.5 lM
CuSO4, 22.5 lM MnSO4, 0.093 lM (NH4)7Mo6O24,
2.28 lM ZnSO4, and 8 lM Fe-EDTA. The entire nutrient
solution was changed twice a week, and the plants were
cultivated for 3 months in a growth chamber under the
following conditions: photoperiod, 16 h light/8 h darkness;
photosynthetic active radiation, 306–308 lmols s-1 m-2;
day/night temperature, 25/17�C; day/night relative humid-
ity, 70/80%.
J Plant Res
123
Half of the plants then received nutrient solution sup-
plemented with 200 lM Al (Al3? activity, 111 lM,
according to GEOCHEM speciation), while the rest of
the plants continued to receive the control solution.
After 2 weeks of treatment, fully expanded leaves (aged
approximately 14 weeks) were used for the analyses.
Bulk element analysis
After harvesting, the tea leaves were washed with distilled
water and oven dried for 48 h (90�C). This dried material
was wet digested in a mixture of acids (69% HNO3: 30%
H2O2, 5:2 v/v) in closed Teflon vessels, using a microwave
oven (O.I. Analytical, model7295, College Station, TX,
USA). The Al concentrations in these digests were ana-
lyzed by inductively coupled plasma-optical emission
spectroscopy (Polyscan 61E, Thermo Jarrell-Ash Corp.,
Franklin, MA, USA).
Sample preparation for low energy X-ray fluorescence
Since chemical fixation of plant material as well as use of
cryo-protectans leads to element redistribution and mis-
placement, tea leaf samples were prepared using cryofix-
ation (Vogel-Mikus et al. 2008b, 2009). Tea leaves were
cut into small pieces (2 9 5 mm) with a razor blade, and
these were immediately put inside 2-mm-wide stainless
steel needles, embedded with a droplet of tissue freezing
medium (Jung) to provide support during the cutting and
rapidly frozen in liquid propane cooled with liquid nitrogen
(Vogel-Mikus et al. 2008b, 2009). The leaf pieces were
then sectioned with a Leica CM3050 cryo-microtome
(Leica, Bensheim, Germany) with head and chamber
temperatures in the range of -35 to -25�C, at a section
thickness of 20 lm. The sections were placed in pre-cooled
Al holders, and freeze-dried at -30�C and 0.4 mbar pres-
sure for 3 days in an Alpha 2-4 Christ freeze dryer, using a
cryo-transfer assembly that was cooled by liquid nitrogen
(Vogel-Mikus et al. 2008b, 2009). Dry leaf cross-sections
were mounted on gold transmission electron microscopy
grids (SPI Supplies; 100 9 100 folding square mesh). The
images of the cross-sections were obtained with an Axio-
skop 2 MOT microscope (Carl Zeiss, Goettingen, Ger-
many), using visible and blue-light excitation source,
equipped with an Axiocam MRc colour digital camera,
using AxioVision 3.1 software.
Low energy X-ray fluorescence microscopy set-up
The LEXRF measurements were performed using the
TwinMic X-ray fluorescence spectro-microscope (Kaulich
et al. 2006) at the Elettra Synchrotron Radiation Facility in
Trieste (http://www.elettra.trieste.it), (BL 1.1L). The X-ray
microprobe for the TwinMic is formed using diffractive
focusing optics. The lateral resolution that can be achieved
with the TwinMic is dependent on the imaging mode of
0.03–1.00 lm. It is operated in the 280–2,200 eV photon
energy range, and as such the TwinMic accesses the main
low-Z elements (K-edge: Z L0 5–9, L-edge Z L0 10–37) that
are of great interest for bio-related and other applications,
as indicated in Fig. 1.
In the present study, the X-ray microprobe was formed
by a tungsten zone plate (Zoneplates.com, UK; http://www.
zoneplates.com) with a diameter of 320 lm, and an out-
ermost zone width of 50 nm. However, the effective lateral
resolution depends on the X-ray source size and geometric
demagnification, which determines the photon flux that is
required for measuring a sufficiently high signal. Consid-
ering this compromise, which is imposed by the type of
sample under investigation, the present measurements were
obtained with lateral resolution in the order of 1 lm.
The transmission signal was acquired using a fast-
readout, electron-multiplying, CCD camera (Andor Ixon)
coupled to a phosphor-screen-based visible light converting
system, which allowed simultaneous detection of bright-
field or absorption, differential absorption, and differential
phase contrast signals (Gianoncelli et al. 2006; Morrison
et al. 2006). The morphological analysis of the specimens
at the cellular and sub-cellular levels was complemented by
a LEXRF set-up consisting of four large-area, Si-drift
detectors (PNSensor, Germany) in an annular back-scat-
tering configuration positioned around the specimen
(Gianoncelli et al. 2009), and a customized preamplifier
and bias electronics established in collaboration with the
Politecnico Milano/National Institute of Nuclear Physics
(INFN), Italy (Niculae et al. 2006; Alberti et al. 2009). The
Fig. 1 Characteristic X-ray fluorescence energy of elements versus
atomic numbers. Elements accessible by the TwinMic set-up at the
Synchrotron Elettra (Trieste) are highlighted in the shaded rectangle(redrawn after Kaulich et al. 2009)
J Plant Res
123
lateral resolution achieved is currently limited by the
necessary signal-to-noise ratio of acquisition. The system is
operated from the boron to the phosphorus K-absorption
edges, with a measured detection limit of about 10 ppm for
the K-absorption edges (Kaulich et al. 2009).
Two regions of the 20-lm-thick, Al-treated leaf cuttings
were scanned as 80 9 80 lm2 (80 9 80 pixels): epider-
mis–mesophyll and xylem–phloem (Fig. 2). This was done
first in scanning transmission mode with a dwell time of
40 ms per pixel. In LEXRF mode, the selected regions
were scanned first with 1.7 keV excitation energy to reach
the Al–K absorption edge, with 10 s dwell time per pixel.
The same regions (80 9 80 lm2, 80 9 80 pixels) were
then scanned again with 2.2 keV excitation energy to reach
the P–K absorption edge, with 11 s dwell time per pixel.
The XRF spectra obtained were fitted using the PyMCA
XRF data analysis software (Sole et al. 2007), with the
application of the MCA Hypermath algorithm and a con-
stant baseline correction. In the results only the represen-
tative element localization maps of three scans per selected
region are presented.
Colocalization analysis
Colocalization analysis of Al intensities with other elements
measured using LEXRF was performed by ImageJ program
using plug-in ‘‘Intensity correlation analysis’’, generat-
ing Pearson’s correlation coefficients (r) (http://www.
macbiophotonics.ca/imagej/colour_analysis.htm). Pearson’s
correlation coefficients range from 1 to -1, where a value of
1 represents perfect correlation/colocalization; value of -1
represents perfect exclusion, and zero represents random
localization.
Results and discussion
The hydroponically cultivated tea plants showed vigorous
growth under both control and Al-treated conditions. The
plants with the high Al3? activity in the nutrient solution
did not show any toxicity symptoms during the 2 weeks
exposure time. Root growth was rather stimulated than
inhibited by the Al treatment (data not shown). This
behaviour confirms the extraordinary high Al tolerance of
tea plants (Matsumoto et al. 1976), which is in contrast
with other plant species where a 50% reduction in root
growth can be caused by Al3? activities between 1 and
25 lM (Barcelo et al. 1996; Poschenrieder et al. 2008).
Under field conditions, tea leaves progressively accu-
mulate Al throughout their life. Al preferentially accumu-
lates in old leaves, but not in young leaves. The ratio of Al
concentration in old to young leaves varies from 18- to
51-fold depending on the growth conditions (Matsumoto
et al. 1976). In bushes of several decades of age, leaves that
are two or more years old have been shown to contain
20–30 mg g-1 Al on a dry weight basis (Matsumoto et al.
1976; Fung et al. 2009). In the present study, the much
younger, but fully expanded, leaves of young plants treated
with the Al for only 2 weeks had much lower Al concen-
trations: on average, 1.0 ± 0.2 mg g-1 dry weight. This is
within the range of values previously reported for young
leaves from soil-grown tea seedlings, and it is in contrast to
the concentrations reported in young leaves of old tea
bushes (Fung et al. 2009).
Localization of the elements in these tea-leaf cross-
sections using LEXRF showed that Al was mainly local-
ized in the cell walls of the epidermal cells (Fig. 3). A
Fig. 2 Tea-plant leaf cross-section (20 lm thick) photographed
under light stereo-microscope (upper) and light microscope using
blue light excitation source (lower) with the indicated scanned regions
of the epidermis–mesophyll and xylem–phloem. The size of the
scanned regions was 80 9 80 lm2
J Plant Res
123
similar distribution was reported in previous studies using
EDXMA on freeze-fractured leaf samples (Carr et al.
2003). Karley et al. (2000) suggested that ions that are not
preferentially taken up by the bundle sheath plasma
membrane move from the leaf vascular tissues via vein
extensions to the epidermis, resulting in the deposition of
excess and non-required metals in epidermal tissues.
Typical accumulation of metals in epidermal cells can be
seen in Ni, Zn and Cd hyperaccumulating plants (Kramer
et al. 1997; Frey et al. 2000), where the metals are mainly
accumulated in the vacuoles of epidermal cells, and only to
a lesser extent in the cell walls (Vazquez et al. 1992, 1994;
Frey et al. 2000; Vogel-Mikus et al. 2008a, b). Accumu-
lation of excess metals in epidermal cells without photo-
synthetic activity is considered a common mechanism for
metal tolerance in hyperaccumulating plants. However,
excess metals seem to be specifically excluded from the
vacuoles of the chloroplast-containing guard cells (Frey
et al. 2000). In fact, stomata are extremely sensitive
to excess metal ions, as has been shown for Al (Schnabl
and Ziegler 1975) and other toxic metals (Barcelo and
Poschenrieder 1990).
In the mesophyll cells of the tea leaves examined here,
only a weak Al signal was observed from the cell walls,
while in the selected leaf regions, no Al signal was detected
from the mesophyll symplast. The cell walls of the epi-
dermis had much lower Si and P contents, when comparing
to the mesophyll cells (Fig. 3), indicating that these ele-
ments have no role in Al sequestration. Si was probably
present in the plant tissues mainly from the seed reserves,
since no Si was intentionally added to the nutrient solution.
Despite low Si concentrations present in leaf tissues, it can
be assumed from the localization maps (Fig. 3c–g), as well
as colocalization numerical analysis (Table 1) that Al
probably displaces Si from negatively charged cell-wall
components e.g. free pectin residues, when Si is present in
leaf tissues at low concentrations (Yang et al. 2008). This is
in contrast to observations in Faramea marginata Cham.
(Rubiaceae) (Britez et al. 2002), where a positive correla-
tion between Al and Si levels was seen for the stems and
leaves. Similar spatial distributions of Al and Si have been
reported for conifers (Hodson and Sangster 1999) and for
the shoots of F. marginata, leading it to be concluded that
co-deposition of Al and Si may substantially contribute to
the internal detoxification of Al (Britez et al. 2002). In
buckwheat roots, complexation of Al to P was seen, which
might also contribute to Al detoxification, since Al–P
complexes formed in the cell wall can prevent Al uptake to
the symplast (Zheng et al. 2005), however in the examined
tea leaves no such colocalization was observed (Fig. 3c–h;
Table 1).
In tea leaves, Al has been proposed to be mainly com-
plexed with catechins and phenolic and organic acids
(Nagata et al. 1992). The catechins content in tea leaves is
usually [15% dry weight and the highest Al content
reported is 31 mg g-1, so the amount of catechins is suf-
ficient to chelate these large amounts of Al (Nagata et al.
Fig. 3 Scan of the representative epidermis–mesophyll region of a
tea leaf (size 80 9 80 lm2, scan 80 9 80 pixels). a Bright field
(absorption) image; b differential phase-contrast image; LEXRF maps
of c carbon; d oxygen; e magnesium; f aluminium; g silicon; and
h phosphorus. The C, O, Mg and Al maps were obtained after
scanning with the 1.7 keV beam, while the Si and P maps were
obtained after scanning with the 2.2 keV beam. The maps were
normalized to the intensity of the beam current and the time of
acquisition (counts s-1 mA-1)
Table 1 Pearson’s (r) correlation coefficients between Al and other
element intensities measured in tea leaves using LEXRF
Correlation Whole epidermal/mesophyll region Epidermal region
r r
Al–C 0.15 0.64
Al–O 0.37 0.65
Al–Mg 0.28 0.65
Al–Si -0.21 0.16
Al–P -0.18 0.11
Intensity correlation analysis was performed using ‘‘Image J’’ pro-
gram. Zero–zero pixels are not included in this calculation. Channel
threshold (1; 255; P \ 0.05)
J Plant Res
123
1992). However, studies of the localization of catechins in
tea-leaf tissues have been contradictory. Suzuki et al.
(2003) showed that catechins are mainly localized in the
vacuoles of mesophyll cells, and not in epidermal cells.
Recent studies using vanillin-HCl staining have reported
the location of catechins in young tea leaves in the palisade
parenchyma cells, vascular bundles, chloroplasts of meso-
phyll cells, vessel walls, and only to a lesser extent, in the
epidermis (Liu et al. 2009).
The intense Al localization in the cell walls of epidermal
cells in our experimental plants confirms the view of
Matsumoto et al. (1976) that Al is mainly moved through
the leaf apoplastically, together with the water mass flow
from the xylem to the epidermis, where the water then
evaporates into the air and Al is concentrated in the cell
walls at the sites of evaporation. This is also supported by
the observations of Carr et al. (2003), who found remark-
able concentrations of Al especially in outer epidermal cell
walls, and also in the cuticle. In buckwheat, for example, it
has been shown that the Al distribution in leaves is con-
trolled by the rate and duration of transpiration, and that Al
is not mobile once it has accumulated in the leaf (Shen and
Ma 2001). In contrast to tea, however, buckwheat seems to
store excess Al mainly in the leaf-cell vacuoles (Shen et al.
2002), as an Al-oxalate complex. Direct measurements of
aluminium uptake and distribution in single cells of Chara
corallina showed that the cell walls are the major site of Al
accumulation; nonetheless membrane transport occurs
within minutes of exposure and is supported by subsequent
sequestration in the vacuole (Taylor et al. 2000). This is
mainly connected to the higher binding affinity of Al3? to
negatively charged hydroxyl and carboxyl groups, conse-
quentially substituting other cations from the cell wall
components, such as for example Ca2? (Yang et al. 2008),
which is supported also by a high degree of Al and oxygen
colocalization (Table 1). It can be also assumed that epi-
dermal cell walls of tea leaves can have different propor-
tions of pectin and hemicellulose when compared to the
mesophyll cell walls, similarly as suggested for root tips of
Al resistant and non-resistant rice varieties, which con-
tained different proportions of pectin and hemicellulose
and consequentially bound different amounts of aluminium
(Yang et al. 2008). However, further studies of Al transport
and compartmentation within the leaves of tea plants and
thorough investigations of the composition of epidermal
and mesophyll cell walls are needed for any absolute
conclusions to be made here.
In the vascular region of the leaf, Al was more or less
evenly distributed. Surprisingly, however, a slightly higher
Al signal was seen for the phloem region (Fig. 4).
Although this had considerably lower signal intensity than
in the epidermal cell walls, the location of Al in the
phloem region indicates that some of the Al can move
symplastically through the leaves. These data suggest that
part of the Al reaching the leaves by xylem transport is
retrieved from the transpiration stream by lateral export to
the phloem tissue. No co-localization was seen with P or
the other elements measured here, which is in line with
reports on Al transport via the xylem as a complex bound
to organic acids, such as citrate in tea plants (Morita et al.
2004). More than 20 years ago, simple haematoxylin
staining procedures had already shown the specific location
of Al in the phloem tissue of Al-accumulating species from
the Brazilian Cerrado region (Haridasan et al. 1986). Here,
this specific localization of Al in the phloem tissue is thus
finally confirmed in the Al-accumulating tea plant using a
sample preparation technique that avoids retranslocation of
the elements during the sample preparation procedure.
In conclusion, LEXRF is a powerful tool for reliable
localization of Al in plant tissues with at least an order of
magnitude higher sensitivity when comparing to EDXMA.
Fig. 4 Scan of the representative xylem–phloem region of a tea leaf
(size 80 9 80 lm2, scan 80 9 80 pixels). a Bright field (absorption)
image; b differential phase contrast image; LEXRF maps of c carbon;
d oxygen; e magnesium; f aluminium; g silicon; and h phosphorus. C,
O, Mg and Al maps were obtained after scanning with the 1.7 keV
beam, while the Si and P maps were obtained after scanning with the
2.2 keV beam. The maps were normalized to the intensity of the beam
current and the time of acquisition (counts s-1 mA-1)
J Plant Res
123
Using LEXRF we confirmed that in young, fully expanded
tea leaves, the preferential deposition of high Al concen-
trations occurs at the end of the transpiration stream, in the
epidermal cell walls. However, the unequivocal localiza-
tion of Al in phloem tissue in the present study also gives
strong support to the view that in Al-accumulating plants,
some Al also can be retrieved from the transpiration stream
and transferred to the phloem.
Acknowledgments The work was supported by the Spanish
(BFU2007-60332/BFI) and Slovenian Government PO-0212, Biology
of Plants Research Programme, and the EU COST 859 entitled
‘‘Phytotechnologies to promote sustainable land use and improve food
safety’’. We thank Maya Kiskinova from Elettra–Sincrotrone Trieste
for her support and for helpful discussions, Antonio Longoni from
Politecno Milano/INFN and Roberto Alberti from XGLab, for their
support in the installation and operation of the LEXRF detector
system.
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