Annals of Botany 103: 665 –672, 2009
doi:10.1093/aob/mcn264, available online at www.aob.oxfordjournals.org
BOTANICAL BRIEFING
Using synchrotron X-ray fluorescence microprobes in the study of metal
homeostasis in plants
Tracy Punshon1,*, Mary Lou Guerinot1 and Antonio Lanzirotti2
1
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA and 2Consortium for Advanced Radiation
Sciences, University of Chicago, Chicago, IL 60637, USA
Received: 4 August 2008 Returned for revision: 27 August 2008 Accepted: 8 December 2008 Published electronically: 31 January 2009
† Background and Aims This Botanical Briefing reviews the application of synchrotron X-ray fluorescence
(SXRF) microprobes to the plant sciences; how the technique has expanded our knowledge of metal(loid)
homeostasis, and how it can be used in the future.
† Scope The use of SXRF microspectroscopy and microtomography in research on metal homeostasis in plants is
reviewed. The potential use of SXRF as part of the ionomics toolbox, where it is able to provide fundamental
information on the way that plants control metal homeostasis, is recommended.
† Conclusions SXRF is one of the few techniques capable of providing spatially resolved in-vivo metal abundance
data on a sub-micrometre scale, without the need for chemical fixation, coating, drying or even sectioning of
samples. This gives researchers the ability to uncover mechanisms of plant metal homeostasis that can potentially
be obscured by the artefacts of sample preparation. Further, new generation synchrotrons with smaller beam sizes
and more sensitive detection systems will allow for the imaging of metal distribution within single living plant
cells. Even greater advances in our understanding of metal homeostasis in plants can be gained by overcoming
some of the practical boundaries that exist in the use of SXRF analysis.
Key words: Metal homeostasis, synchrotron X-ray fluorescence, SXRF, microspectroscopy, microtomography,
X-ray absorption spectroscopy, XAS, ionomics, Arabidopsis thaliana, hyperaccumulator.
IN T RO DU C T IO N
The synchrotron X-ray fluorescence (SXRF) microprobe is a
non-destructive technique for collecting spatially resolved
metal(loid) (hereafter, metal) abundance data, and is ideal
for answering fundamental questions about metal homeostasis
in plants. SXRF collects data on multiple metals simultaneously without destroying the sample. Sample preservation
and sectioning are usually unnecessary. We believe that with
continued advances in X-ray optics and detector sensitivity,
high-resolution three-dimensional imaging of the elemental
composition of a single living cell will become routine. In
this review, we summarize how SXRF has been used, and
how it is developing to meet the needs of the plant science
community. For detailed technical explanations of SXRF and
the related techniques described here, readers are directed to
Sutton et al. (2002).
In this article, metal homeostasis refers to the mechanisms
and control (genetic and environmental) of the uptake, transport and storage of essential and non-essential metals by
plants. Metals are mobilized from the rhizosphere and transported across membranes within the plant before being
stored or assimilated. Therefore, their concentration, location
and chemical form provide information about the pathway
and destination of a particular metal within the plant, and
can be used to infer a metabolic role for the metal. The ultimate aims of metal homeostasis research include improving
the nutritional value of food crops by engineering plants that
take up higher concentrations of nutrient metals into the
* For correspondence. E-mail [email protected]
edible parts (biofortification; Zhu et al., 2007; Mayer et al.,
2008). It is also necessary for engineering crops with the
ability preferentially to exclude or accumulate potentially
toxic elements (Scheckel et al., 2007; Tappero et al., 2007).
Studies presenting spatially resolved metal data in plants
support the idea that the distribution provides information
about homeostatic mechanisms (McNear et al., 2005; Isaure
et al., 2006). For instance, McNear et al. (2005) found that
the Ni hyperaccumulator Alyssum murale stored Ni in the
base of leaf trichomes and epidermal cells, basically sequestering Ni in non-metabolically active cells to prevent toxicity.
Spatially resolved metal abundance data have been used in
studies of the metabolism of numerous metal hyperaccumulator plants (Sarret et al., 2002; Chen et al., 2003, 2005; McNear
et al., 2005; Onuma et al., 2005; Tongbin et al., 2005; Isaure
et al., 2006; Kashiwabara et al., 2006; Scheckel et al., 2007;
Tappero et al., 2007). Metal localization characteristics can
reveal the plant organ in which genes are expressed, and
confirm the function of transport proteins and metallochaperones responsible for moving metals across membranes and
through the cell. Understanding transport mechanisms is critical because transport underpins both uptake and storage.
Silencing or deleting genes that encode metal transport
proteins can dramatically disrupt normal metal distribution,
allowing researchers to infer gene function (Kim et al., 2006).
Other complementary techniques can be used alongside
SXRF to investigate more fully metal homeostasis in plants.
Most commonly, X-ray absorption spectroscopy (XAS) can
be used to provide element-specific, spatially resolved information about metal valance and speciation, even at mg kg21
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Punshon et al. — SXRF and metal homeostasis in plants
abundances. Using XAS to distinguish between organic – metal
complexes in plants remains a challenge, however, owing to the
low metal concentrations coupled with the weak scattering at
extended energies. For in-situ studies, obtaining absorption
spectra with sufficient resolution can be challenging (Isaure
et al., 2006). Spatially resolved XAS is most successfully
applied in probing the valance states for metals such as Cr
(Howe et al., 2003) and metalloids such as As and Se
(Pickering et al., 1995, 2006) because these data are much
easier to evaluate conclusively even at very low concentrations.
There are unanswered questions at every level of metal
homeostasis that can benefit from the use of SXRF microspectroscopy: from mobilization from the soil and uptake of metal
ions by the root to the transporters involved in xylem loading
and unloading, trafficking and storage (Clemens et al., 2002).
With the exception of Fe, which is comparatively well characterized, little is known about the mobilization of trace elements
by plant roots. Characterizing the genes responsible for metal
ion homeostasis is a major area of scientific endeavour because
it is the key to the manipulation of metal levels via numerous
molecular breeding techniques, and we believe SXRF has particular utility here. The mineral nutrient and trace element
composition of an organism is known as the ionome (Salt,
2004), representing the inorganic components of the cell and
the organism as a whole (Salt et al., 2008). The systematic
approach to characterizing the entire complement of metal
homeostasis genes involves collecting the elemental profiles
(metal concentration of root and shoot material) of natural
populations (ecotypes), mutants and transgenic lines of the
model plant Arabidopsis thaliana grown from seed under standardized conditions, and making this information widely available in a searchable database (www.purdue.edu/dp/ionomics/)
(Baxter et al., 2007). The database is a valuable resource for
the unbiased identification of candidate metal homeostasis
genes. The database can be queried by metal, and lines with
levels that deviate significantly from the wild-type are returned
in the search. The elemental profile is determined by use of
inductively coupled plasma mass spectroscopy (ICP-MS), a
volume-averaged technique that provides excellent sensitivity,
accuracy and precision. Nonetheless, volume-averaged methods
provide a limited view of homeostasis mechanisms (Salt, 2004).
Incorporating spatially resolved data into the database would
accelerate the rate at which functional information was assigned
to genes and would generate new hypotheses. Ionomic imaging
would allow co-localization of in-vivo gene expression and
protein localization patterns with ionomic changes, providing
spatial linkage between gene, protein and ionomic function
(Salt, 2004). SXRF is not a high-throughput technique,
however; screening for metal phenotypes is not a practical
use of the technology. High-resolution ICP-MS is able to
detect ultra-low concentrations of multiple metals rapidly,
often achieving detection limits in the high pg kg21 range, so
it is the technique of choice for initial screening. One can
then use SXRF to image the metal distribution in a range of
mutants with altered metal distribution for comparison with
wild-type plants.
Experimental challenges remain in making SXRF imaging a
more accessible tool for the plant sciences. Firstly, virtually all
hard X-ray microprobe instruments worldwide are significantly
oversubscribed, and access to resources is highly competitive.
New, more powerful synchrotron facilities are under construction with more beam lines dedicated to X-ray microprobe
analysis of biological materials, but availability is still unlikely
to meet demand in the near future. Efficient methodology and
increased collaboration between research groups would go
some way towards preventing repetition of time-consuming
analyses (for instance, of wild-type plants), and allow
interpretation of the complete multi-element data set, maximizing use of time and data. This kind of collaborative
research is already well established in plant molecular genetics. The development of specialized sample stages that can
accommodate living tissue and maintain tissue hydration
over extended periods will allow in-vivo data to be collected
routinely, and some efforts are underway to develop these.
Development of detectors with enhanced sensitivity and electronics that allow for faster integration, for analysing tissues
with lower (i.e. non-hyperaccumulator) levels of metals over
much shorter periods of time, is currently under way (Ryan
et al., 2007).
Researchers need to be able to see where the metal is at the
level of the whole plant, the individual cell and the organelle.
They need to be able accurately to compare metal concentration and speciation between plants altered with respect to
the genes they express and/or the environmental conditions
under which they were grown. This requires a mm and
sub-mm spatial resolution and a sub-mg kg21 detection sensitivity. This is likely to be more commonly available in the
newest generation of instruments, which use low-emittence
sources, improved focusing optics and multi-element detectors
with large active areas. Analysing biological material without
introducing structural or chemical artefacts at sub-mm resolution is challenging. While SXRF methods in general
deposit much less power on the sample than other microbeam
methods such as electron microprobe analysis (EMA),
particle-induced X-ray emission (PIXE), etc., the effects of
dehydration, preservation and sectioning all need to be evaluated because they may potentially alter metal distribution and
speciation. Even with these caveats, SXRF still provides
researchers with a technique that in many cases requires only
the bare minimum of sample preparation, opening the way
for collection of in-vivo data from intact living plant
specimens.
S X R F I N T H E PL A N T S C I E N C E S
Synchrotron X-ray fluorescence microprobes have been
demonstrated as being potentially useful analytical tools for
plant tissues since the late 1990s, although they have been
under-utilized by the plant science community. Advances in
X-ray focusing optics and solid-state X-ray detectors, many
utilizing arrays of X-ray-sensitive materials, have led to the
development of a new generation of instruments with drastically improved spatial resolution and detection sensitivity.
Current instruments boast sub-mg kg21 detection limits for
transition metals at spatial resolutions of 100 nm. Currently
detection limits in the low-mg kg21 to high-mg kg21 range
and resolutions on the order of 5 – 10 mm are routinely
achieved. The next generation of instruments being planned
will provide spatial resolutions between 1 and 30 nm, which
Punshon et al. — SXRF and metal homeostasis in plants
will allow for a more accurate representation of the metal distribution and abundance in biological samples.
Techniques for abundance calculation (conversion of fluorescence yields to metal concentration on a per weight or per
area basis) vary depending on the nature of the material
being analysed. SXRF is a reference method and standards
are generally required for quantitative results. In its most
basic form, where matrix-matched standards are available
(same thickness, density and bulk composition), the standards
can be analysed, fluorescence intensities obtained and then a
calibration curve of intensity vs. concentration generated.
While this is the most accurate approach, this is also the
most impractical; matrix-matched standards that are spatially
homogenous at the mm scale (both at the surface and at
depth) are scarce. Quantification based on first principles is
feasible, computing the concentration from incident beam
intensity and measured fluorescence intensity while all the
intermediate processes are taken into account, but this is
quite difficult in practice. A fundamental parameters (FP)
approach is very effective when coupled with standards analysis. Here, the relationship between concentration and intensity
is determined with the standards, while FP algorithms are used
to correct for absorption differences between sample and standards. This computation takes into account various parameters
of the analysis conditions (absorption of the X-rays by the Be
windows, the air path between the sample and the detector, fluorescence yields, photoionization efficiencies, self-absorption,
secondary fluorescence) and the matrix composition (thickness, density, major elemental composition). This approach
is built into a number of software packages available at
SXRF facilities and results can be within 5 – 10 % of actual
values. A further advantage is that it is not necessary for the
standard to contain all the elements of interest in the sample.
The FP algorithms can extrapolate predicted fluorescence
yields and absorption effects to other emission lines detected.
McNear et al. (2005), for instance, used a standard-based FP
approach, using National Institute of Standards and
Technology (NIST) thin-film standards to calculate the sensitivity to Ca, Mn, Fe, Cu and Zn (counts per second per mg
cm22). Their study featured a Ni-hyperaccumulating plant,
although the thin-film standard did not contain this element.
They therefore inferred the sensitivity to Ni by interpolating
by atomic number between Fe and Cu.
S X R F A ND S T UD IE S OF M E TA L HO M E OS TA S IS
One of the earliest studies to demonstrate the potential utility
of SXRF microprobes examined the arbuscular mycorrhizal
fungus Glomus mosseae associated with roots of Plantago
lanceolata (Yun et al., 1998). Mycorrhizal association strongly
influences plant metal uptake, reducing or preventing the
uptake of toxic concentrations of metals and allowing plants
to survive in contaminated soils. They are naturally very
useful for contaminated land re-vegetation. Because of the
close association between fungal and plant tissue it is difficult
to determine the distribution and abundance of elements
between the host plant and symbiont in situ. Yun et al.
(1998) collected elemental maps of P, S, K, Ca, Mn, Fe, Ni,
Cu and Zn with a 1 3 mm beam from intact, inoculated
root tissue. This highlighted the possibility of mounting
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hydrated plant samples for analysis by mapping fresh root
samples placed between two layers of Kapton (metal free)
tape (Dupont High Performance Films, Circleville, OH,
USA). This uncovered a strong association between Cu and
Zn and fungal hyphae, allowing these elements to be used as
surrogate measures of hyphal presence. This was also found
by Sarret et al. (2003) using tomato (Solanum lycopersicum)
colonized by Glomus intraradices. Elemental signatures are
useful in SXRF mapping; plant tissue has a strong Ca signature
that allows structural components to be identified, although
studies on mycorrhizal fungi are less common. Yun et al.
(1998) observed that mycorrhizal plants contained comparatively less Mn than non-mycorrhizal plants, and imaged Fe
precipitation on the exterior of the root. They compared phasecontrast images collected from wet-mounted samples with
those that had been prepared using standard techniques for
optical microscopic imaging. The images were very similar,
although the wet-mounted samples showed far more detail in
the stele, which collapses when the root is dried.
Despite early success with hydrated samples, SXRF
imaging studies on plant tissue have continued to use dehydration, resin-embedding and sectioning (Naftel et al., 2001)
or freeze-drying (McNear et al., 2005), with varying degrees
of success. Preservation is often necessary for the experimental
configurations at some beam lines, such as a detector requiring
a vacuum enclosure. Freeze-drying techniques appear to be the
least disruptive way of preserving plant tissues for SXRF
analysis. Hansel et al. (2001) used freeze-drying effectively
to preserve Phalaris arundinacea (reed canary-grass) root
tissues for SXRF microprobe and fluorescence computed
microtomographic imaging (fCMT). They studied As uptake
and found that As is sorbed to Fe plaques on the exterior
root surface. fCMT generates a reconstructed cross-section of
the metal distribution in a whole object by utilizing back projection or fast Fourier transform reconstruction algorithms to
project the fluorescence data on to a 2D reconstructed image,
similar to the way the X-ray attenuation through an object is
reconstructed in a medical CT scan. The fluorescence data
are collected from rastering of the sample horizontally
through the focused beam as a function of angle. The advantages of fCMT include removing the need physically to
section fragile, small or rare samples. Disadvantages lie in
the additional absorption of characteristic X-rays by the
sample itself on their path to the detector system, although corrections can be made to account for self-absorption. For lowenergy X-rays or where samples are particularly dense or
large (exceeding a few hundred micrometres), absorption
effects can be severe and difficult to correct.
Pickering et al. (2000) paired SXRF with spatially resolved
XAS to image the distribution of Se species in the Se hyperaccumulator Astralagus bisulcatus. They tuned the incident
beam to the absorption edges of the two dominant forms of
Se: selenomethionine and selenate. By mapping the same
region of the sample at each energy, they generated overlapping images of the selenomethionine and selenate distribution.
This combination of techniques has proven to be a particularly
informative way to infer homeostasis mechanisms. After
mapping of Se species in mature, intermediate and young
leaf and root tissue, it was found that Se speciation differed
according to the developmental stage of the plant, with a
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Punshon et al. — SXRF and metal homeostasis in plants
predominance of selenomethionine in the roots and young
leaves, and selenate in mature leaves. In a follow-up study,
Pickering et al. (2003) showed that seleno-Cys methyltransferase, the enzyme responsible for selenate biotransformation,
was expressed regardless of Se exposure or tissue age, indicating that developmental limitation in Se biotransformation
occurs at an early stage in the pathway. Freeman et al.
(2006) used SXRF in combination with energy dispersive
X-ray microanalysis, liquid chromatography– mass spectroscopy and XAS to explore the biochemical basis of Se
hyperaccumulation in A. bisulcatus and Stanleya pinnata.
Howe et al. (2003) used SXRF and XAS to study the localization and oxidation state of chromium (Cr) in fresh leaves
of subterranean clover (Trifolium brachycalcinum). They collected elemental maps from fresh leaf tissue of Cr(VI)treated plants immediately prior to analysis, demonstrating
reduction to the less toxic Cr(III). The characteristic reddening
of the leaf corresponded to localized tissue Cr accumulation.
The successful application of XAS to fresh plant tissue
was notable in this study. Schekel et al. (2004) used SXRF
to explore thallium (Tl) distribution in hyperaccumulator
Iberis intermedia, and used XAS to provide information
about Tl speciation. More toxic than mercury or cadmium,
Tl is chemically similar to potassium and is readily taken up
by plants. Leaves of the hyperaccumulator secured to the
XYZ stage were still attached to the plant. Some beam-induced
sample damage was observed after replicated analysis over
the same region of tissue, but this was minimized by reduction
of the dwell time without compromise of data quality. Tl
was present within the vasculature of leaf tissue in a
form similar to monovalent Tl, confirming that I. intermedia
could safely be used to phytomine Tl. The alternative
oxidation state, Tl(III), is 43 000 times more toxic than Cd
on a free-ion basis. Using fCMT, Scheckel et al. (2007)
confirmed the confinement of Tl to the vascular system
by reconstructing a three-dimensional image of the Tl
distribution.
McNear et al. (2005) used fCMT to image the distribution
of Ni species in the hyperaccumulator Alyssum murale
‘Kotodesh’. They were able to determine the pathway of Ni
transport from fine roots, through the xylem to the leaf
dermal tissues, trichome bases and tips. They imaged Ni
accumulation in leaf epidermal cells and exclusion from the
mesophyll cells. Pickering et al. (2006) used XAS imaging
to show the distribution of arsenate [As(V)] and arsenite
[As(III)] in Pteris vittata (Chinese brake fern). This species
X
Point 2
P
S
CI
Cd
K
Ca
Point 1
High
Low
Concentrations
F I G . 1. SXRF elemental maps of the leaf of Arabidopsis thaliana showing Cd enrichment of the trichome in Cd-exposed plants. Scale bar ¼ 70 mm. Beam
dimensions 0.9 mm (horizontal) and 0.3 mm (vertical), using a 1-mm step size. Dwell times 500 ms pixel21. ‘Point 1’ and ‘Point 2’ correspond to regions of
interest from which X-ray absorption spectra were collected. Reproduced from Isaure et al. (2006), with permission.
Punshon et al. — SXRF and metal homeostasis in plants
of fern is one of the few arsenic (As) hyperaccumulators, and
its discovery generated a great deal of interest in the field of
phytoextraction. Mapping showed that As was transported as
As(V) and then reduced to As(III) and stored in the vacuole.
Arsenic was excluded from cell walls, rhizoids and reproductive tissue, preventing metal-induced damage.
Isaure et al. (2006) used SXRF to investigate Cd localization and speciation in Arabidopsis thaliana grown under
Cd-enriched conditions. The SXRF imaging of a large area
of the leaf surface showed clearly that Cd was localized in
the trichomes (Fig. 1). Studies of Cd compartmentalization
in the hyperaccumulator A. halleri implicated the mesophyll
cells as sites of Cd storage (Kupper et al., 2000), but this
was not observed in A. thaliana. The distribution of Ni,
cobalt (Co) and Zn in leaves of the Ni hyperaccumulator
Alyssum murale was investigated by use of SXRF imaging
(Tappero et al., 2007) (Fig. 2). These 2D images show trichomes enriched in Ca, and mesophyll enriched in Ni, Co
and Zn. Cobalt in particular is localized to the leaf tips and
margins. fCMT showed that Ni was localized to the epidermal
layer but Co was excluded from these cells, forming a Co-rich
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mineral precipitate on the leaf surface. Clearly, homeostatic
regulation of Ni and Co differ widely in A. murale in the presence of a mixture of metals.
SXRF and XAS are ideal techniques for studying As in rice
(Meharg et al., 2008). The chronic poisoning of people in
Bangladesh and West Bengal as a result of drinking
As-contaminated well water is an unprecedented human
health crisis; almost 40 million people are at risk from As
exposure (Nordstrom, 2002). Conservative estimates hold
that irrigation of rice crops with contaminated well water in
Bangladesh during the dry season could raise the soil As concentration by 1 mg g21 per annum (Meharg and Rahman,
2003), elevating As concentrations in rice grain. A market
basket survey of rice from various countries uncovered elevated As in US rice, although it is speciated differently from
that in rice grown in Bangladesh (Williams et al., 2007).
Dietary studies show that even in the West, rice consumption
contributes significantly to As dietary intake. It is now
thought that rice, in particular, accumulates As as a side-effect
of a physiological demand for silica (Ma et al., 2008). Such
findings prompted work on the distribution and speciation of
Ni
Ca
Zn
Ca
Co
Ca
F I G . 2. SXRF two-colour images of Ni, Co and Zn distribution in a hydrated leaf of the hyperaccumulator Alyssum murale treated with a mixture of Ni, Co and
Zn. The camera image shows the region of the leaf selected for imaging. Reproduced from Tappero et al. (2007), with permission.
Punshon et al. — SXRF and metal homeostasis in plants
A
D
416
116
100
355
128
61
0
0
0
0
0
0
mg kg–1
670
Mn
Fe
Zn
Fe
Mn
Zn
B
Col-0
C
vit1-1
E
Col-0
vit1-1
F
G
H
Col-0
vit1-1
F I G . 3. SXRF microtomography of Arabidopsis seed. (A) Light micrograph cross-section of a mature seed. Scale bar ¼ 62 mm. Seed cross-sections show the
radicle (embryonic root; left) and the two cotyledons (embryonic leaves pressed together; larger structure on the right). (B, C) Total X-ray absorption tomographic
slices of Columbia-0 (wild-type) and vit1-1 mutant seeds. Scale bar ¼ 100 mm. (D) SXRF tomographic slices of Fe Ka (blue), Mn Ka (green) and Zn Ka (red)
fluorescence lines collected from Columbia-0 and vit1-1 seeds with metal abundances indicated in mg kg21 (smaller images), and composite images of Fe, Mn
and Zn abundance of Columbia-0 and vit1-1 (larger images). (E) Three-dimensional rendering of total X-ray absorption of a wild-type Arabidopsis seed. (F)
In-silico sectioned (y axis, upper 50 % removed) rendering of the total X-ray absorption image shown in (E). (G, H) Three-dimensional rendering of Fe Ka
X-ray fluorescence in Columbia-0 and vit1-1, respectively, with both seeds identically orientated.
As in rice. Using SXRF and laser ablation interfaced with
ICP-MS, Meharg et al. (2008) found differences between
polished and unpolished rice for both the distribution and
the speciation of As. Arsenic is found distributed relatively
evenly in the endosperm of polished rice, although grains
with an intact pericarp and aleurone layer (unpolished brown
rice) had much higher As concentrations in these tissues.
Meharg et al. (2008) explain As localization in the aleurone
layer as resulting from granules of phytic acid (inositol hexakisphosphate), since these are also rich in Ca, Mg, K and Zn,
with which As co-localizes.
Recently, 2D SXRF elemental mapping, fCMT and XAS
were used to illustrate how mature compost could be used to
reduce Zn uptake by Eruca vesicaria growing in contaminated
soil. Plants grown in compost-amended soils were partially
able to block Zn uptake at the root endodermis by complexing
it as the insoluble Zn phytate, with a much smaller fraction
gaining entry to the vascular system as Zn citrate (Terzano
et al., 2008).
SXRF also has great potential as a tool in molecular genetics
research to help characterize the function of genes involved in
metal ion homeostasis. Young et al. (2006) used SXRF to
screen a large array of seeds from transgenic lines of the
model plant Arabidopsis thaliana. This study used a large
unfocused beam (500 500 mm) to collect low-resolution
2D elemental maps. Kim et al. (2006) realized the potential
application of SXRF to molecular genetics when they used
SXRF and fCMT in combination with established genomic
techniques to characterize the function of the gene AtVIT1
(vacuolar iron transporter 1) in A. thaliana seed. The plant
vacuole is a key storage location for Fe, and VIT1 is an Fe
transporter found on the vacuolar membrane. A threedimensional rendering of the Fe Ka fluorescence from dry,
un-sectioned seed showed that wild-type plants localize Fe
in the layer of cells surrounding the vasculature of the entire
embryo (Fig. 3G, H). [The Ka fluorescence line results from
electron transition to the innermost ‘K’ shell from a 2p
orbital of the second, or ‘L’ shell. The Ka spectral line is
the strongest spectral line for an element.] Localization of
VIT1 expression via GFP (green fluorescent protein) fusion
showed it to be expressed in the vasculature in a similar configuration. Deletion of VIT1 abolished this Fe distribution,
resulting in an altered pattern resembling the characteristics
of Mn distribution (Fig. 3D), with localization to the abaxial
Punshon et al. — SXRF and metal homeostasis in plants
epidermis of the cotyledons and various cortical cells of the
radicle. Because VIT1 is not involved in Fe loading of the
seed as originally hypothesized, Fe concentration of bulk
seed determined by volume-averaged methods revealed no
difference between wild-type seed and seed in which VIT1
had been deleted. fCMT imaging showed that VIT1 was
involved in localization of Fe to cells surrounding the embryonic vasculature, presumably in preparation for rapid growth
during germination. The wider aims of this research were to
understand the control of Fe loading of the seed as a way of
biofortifying crop plants, to address nutritional Fe deficiency.
These studies were complementary to those carried out on
the nramp3nramp4 double mutant, in which electron
microscopy was used to image Fe associated with globoids
within the vacuole (Thomine et al., 2003; Lanquar et al.,
2005).
For the most part, plant scientists have used SXRF as a
means of answering questions of where the metal is located
in the plant, and then used this information to make broad
inferences about the mechanisms responsible. However,
when SXRF is used as a tool in molecular genetics alongside
methods that show the location of gene expression, it can
answer far more specific questions about gene function and
expression. SXRF has emerged as a powerful technique for
plants because it has the potential to provide in-vivo data.
The value of in-vivo data, unencumbered by artefacts of
sample preparation cannot be overemphasized. As we move
ahead with the construction of another generation of synchrotron facilities, capable of using a sub-mm beam to image an
individual cell, maintaining cellular integrity as close to life
as possible will be both a significant challenge and a major
accomplishment.
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