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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