NMR Plats

The Plant Journal (2012) 70, 129146
doi: 10.1111/j.1365-313X.2012.04927.x
HIGH-RESOLUTION MEASUREMENTS IN PLANT BIOLOGY
Surveying the plants world by magnetic resonance imaging

Ljudmilla Borisjuk1,*, Hardy Rolletschek1 and Thomas Neuberger2,3
Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrae 3, Gatersleben, Germany,
2
Department of Bioengineering, Pennsylvania State University, University Park, PA 16802, USA, and
3
Huck Institutes of the Life Sciences, High Field MRI Facility, Pennsylvania State University, University Park, PA 16802, USA
Received 19 October 2011; revised 6 January 2012; accepted 16 January 2012.

*
For correspondence (e-mail borysyuk@ipk-gatersleben.de).
SUMMARY
Understanding the way in which plants develop, grow and interact with their environment requires tools
capable of a high degree of both spatial and temporal resolution. Magnetic resonance imaging (MRI), a
technique which is able to visualize internal structures and metabolites, has the great virtue that it is
non-invasive and therefore has the potential to monitor physiological processes occurring in vivo. The major
aim of this review is to attract plant biologists to MRI by explaining its advantages and wide range of possible
applications for solving outstanding issues in plant science. We discuss the challenges and opportunities of
MRI in the study of plant physiology and development, plantenvironment interactions, biodiversity, gene
functions and metabolism. Overall, it is our view that the potential benefit of harnessing MRI for plant research
purposes is hard to overrate.
Keywords: MRI, seed, non-invasive imaging, plant metabolism, abiotic/biotic stress, biodiversity.
INTRODUCTION
Magnetic resonance (MR) images derive from spatially
encoded nuclear magnetic resonance (NMR) signals. The
first MR images (at that time better known as zeugmatograms) were acquired less than 40 years ago by Lauterbur,
(1973). The non-invasiveness of MRI has encouraged its
widespread adoption and continuing development as a
clinical tool (Simon and Mattson, 1996), and its value was
recognized by the scientific community in the awarding of
the Nobel prize for physiology or medicine in 2003 to its
inventors, Lauterbur and Mansfield.
The earliest application of MRI in the plant sciences was
based on the use of a clinical human NMR scanner (Hinshaw
et al., 1979; Bottomley et al., 1986), but with the size of the
capital expenditure needed to equip and maintain a dedicated MRI facility, along with the rather modest spatial
resolution achieved at the time, optical microscopy maintained its role as the primary means of exploring the internal
structures of plants. At the same time, due to a number of
technical issues specific to plants in particular the wide
diversity with respect to organism size and their sessile
nature plants probably did not represent an attractive
subject for NMR scientists. With the advances in hardware
development in the last decades, the realization of ultra high
2012 The Authors
The Plant Journal 2012 Blackwell Publishing Ltd
magnetic fields, and the development of new imaging

techniques, most of these problems have been solved, and
the way has been paved for the application of MRI in plant
research. Currently, however, the technique remains surprisingly underused, probably as a result of a widespread
lack of awareness of its potential for solving outstanding
issues in plant physiology. This review aims to highlight the
current potential of MRI for applications in plant science, and
also to provide a forward look at likely future developments
in the technique.
WHY USE MRI?
The principles by which MRI images are acquired differ
fundamentally from those underlying conventional optical
methods (Callaghan, 1993). The primary advantage of MRI
is that both static and dynamic parameters can be spatially
resolved, but importantly, the technique generates data in a
non-destructive manner from the interior of the sample. In
this way, the morphology/anatomy of opaque samples of
whatever size, form or composition can be imaged, while at
the same time allowing an assessment of a range of
chemical parameters. Hence, this enables the visualization
of the long-term dynamic behaviour of living plant tissue. It
129
130 Ljudmilla Borisjuk et al.

is possible to generate metabolic maps of the living plant
body, and to use such a data to monitor various physiological processes. As an example, an MRI scan of a living
plant stem can demonstrate the location of the xylem vessels, give information as to whether or not they are filled
with liquid, derive the velocity and direction of this liquids
movement and determine the identity of the metabolites
dissolved in it (Metzler et al., 1995; Van As et al., 2009;
Windt et al., 2009).
Few, if any, other analytical techniques addressing the
physiology and development of living plants in their natural
environment are as versatile as MRI (Ratcliffe, 2010). The
other methods considered for this type of research are based
on either visible range radiation (confocal laser scanning
microscopy, optical coherence microscopy and optical projection tomography), on X-rays (high-resolution computed
tomography) or on positrons (positron emission tomography, PET). A disadvantage of all optical techniques is that
thick specimens usually need to be cleared with an organic
solvent, precluding the possibility of live imaging (Lee et al.,
2006). X-ray irradiation is incompatible with metabolite
analysis, while the spatial resolution of PET is at best
modest. There are a number of excellent techniques like
Raman spectroscopy, matrix-assisted laser desorption/ionization (MALDI) and related mass spectrometry methods
which provide very detailed chemical information on a
spatial scale. However, their use is restricted to either
surface imaging or tissue sections. In contrast, MRI can
image in real time irrespective of sample thickness (Holbrook et al., 2001), and allows the measurement of both the
distribution and dynamics of water and a range of plant
metabolites.
WHAT PRIOR KNOWLEDGE IS NEEDED TO
UNDERSTAND MRI?
Basics
A brief explanation of the physics underlying MRI is given
here, but in the interests of clarity for the non-specialist,
more detailed information has been included in the Supporting Information (Data S1). Readers who are interested
in more details are referred to a number of excellent textbooks. A comprehensive introduction to NMR has been
published by Levitt (2008). A very detailed overview of the
principles of MRI and the most common imaging techniques in medical imaging are described in Haacke et al.
(1999). As in many plant MRI experiments a very high
resolution is of essence; the book by Callaghan (1993) about
MRI microscopy is of particular interest. An excellent and
very detailed essay about radio frequency (RF) resonator
design and construction can be found in Mispelter et al.
(2006). And finally an open access internet book by Hornak
(The basics of MRI, http://www.cis.rit.edu/htbooks/mri/) is
recommended.
(a)
(c)
(b)
(d)
Figure 1. Principles of magnetic resonance imaging (MRI).

(a) Experimental setup for MRI experiments. A carrot, as an example for the
biological subject, is positioned in the centre of the MRI instrument. The lower
part shows the orientations of the existing magnetic fields.
(b) Typical gradient echo pulse sequence applied during an MRI experiment.
(c, d) The MRI of a carrot generates first an image in Fourier (k-)space, which
then has to be converted into image-space, showing the carrot segment
analysed here.
An MRI system is designed to generate three different

magnetic fields: the first (B0) is established by a large static
magnet, the second (Gx, Gy, Gz) by a gradient coil set which
generates three switchable spatially varying orthogonal
magnetic fields, and the third (B1) by a RF resonator which
provides a temporal varying magnetic field orthogonal to
B0 (Figure 1a). To perform a MRI experiment the specimen to
be examined is placed inside the strong magnet with the
static magnetic field B0 always oriented in the z-direction.
Besides permanent and electromagnets, superconducting
magnets with field strengths up to 21 tesla (T) are used.
Magnetic resonance imaging detects atoms having a nonzero nuclear magnetic moment (called spin). The most
commonly used in vivo nuclei are 1H, 13C, 19F, 23Na, 31P and
39
K (see Data S1). While nuclei with a spin of 3/2, for
example, create additional possibilities for imaging, but play
only a minor role, the following is concentrated on nuclei
with a spin of 1/2. Furthermore, as living tissues have a high
concentration of water and the majority of MRI images are
images generated from protons within the water molecule,
MRI of the protons within the water molecule will be
discussed. This charged particle, the proton, has a spin 1/2
with an angular momentum. In the absence of a magnetic
field the spins are oriented randomly, yielding an isotropic
distribution. The net magnetization Mz a sum of all magnetic
moments is zero. If a magnetic field B0 is applied the spins
start to precess with frequency x, called the Larmor
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The Plant Journal 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 129146
Surveying the plants world by MRI 131

frequency. The spin magnetic moment of each proton
moves on a randomly oriented cone. The angle between
the spin magnetic moment and B0 stays constant and the net
magnetization Mz is still zero. Very small fluctuating magnetic fields resulting from the surrounding of the protons
cause a slight change of the angle between the spin
magnetic moment and B0. To reach the most energetically
favourable state with the lowest magnetic energy the
magnetic moments become slightly more oriented towards
the external B0 field resulting in a non-zero net magnetization Mz. Once the lowest energetic state is reached the net
magnetization Mz reaches its maximum value, called the
equilibrium magnetization M0. The frequency of precession
x is proportional to the magnetic field B0, i.e. x = cB0. The
values of the nucleus-specific constant c (also referred to as
the gyromagnetic ratio) and the resulting ones of x in a
magnetic field of 17.6 T are given in Data S1.
The application of a linear polarized oscillating electromagnetic field (B1) with a duration of sB1 at the Larmor
frequency perpendicular to B0 (referred to as an RF pulse),
rotates the equilibrium magnetization M0 towards the
xy-plane. The rotating xy-component (Mxy) of M0 induces a
voltage in the RF resonator which is then recorded by the
NMR spectrometer. Both the transmission of the B1 field and
the detection of the NMR signal are achieved by either a
single or by multiple RF resonators. To maximize the signal,
these antennae need to be fitted closely around the
specimen. The combination of a large RF resonator with a
small specimen would result in a reduced signal to noise
ratio (SNR).
The decay of the signal after a single excitation pulse over
time due to relaxation effects is referred to as free induction
decay. The predominant relaxation effects relate to spin
spin relaxation (described by a relaxation time constant T2)
and to variation in B0 caused by changes in magnetic
susceptibility within the sample. The combined effects are
described by the time constant T2* (T2* < T2). While there
are ways to reverse the effect of variation in B0, spinspin
relaxation cannot be avoided. Spin-lattice or longitudinal
relaxation is a further relaxation effect, which is characterized by the time constant T1. This process occurs either at the
moment when the sample is first brought into the magnetic
field, or after a RF pulse has rotated M0 towards the xy-plane.
T1 describes the time required for the magnetization to
re-establish its equilibrium value M0 along the z-axis.
Image generation and contrast
Thus far, the acquired signal has no spatial information.
Unlike a light microscopy image which is acquired in image
space, the MRI image is acquired in Fourier space (also
referred to as k-space), and the actual image then has to be
reconstructed via a multidimensional inverse Fourier transformation. The encoding of the MR signal is conducted by
switching the spatially varying magnetic field gradients
(Gx,y,z) in a certain manner as shown in the example of a

gradient echo sequence (Haase et al., 1986) in Figure 1b.
A more detailed description of the encoding procedure is
given in Data S1. An illustration of real k-space data
acquired from a slice through a carrot tap root is shown in
Figure 1c, while Figure 1d shows the reconstructed image
following a two-dimensional (2D) inverse fast Fourier
transformation.
Unlike light microscopy images, MRI images are monochromatic, although colour coding can be added. Differences in grey-scale contrast reflect variation arising from
three main sources. The one which the user cannot modify
is the spin density within a given region of the specimen.
Localized high water content, for example, results in
an enhanced signal intensity. The other two sources are
based on the relaxation times T1 and T2/T2*. Different
tissue types within a plant can have different relaxation
times. By adjusting the acquisition parameters of the
pulse sequence a different contrast can be achieved (see
Data S1).
Image resolution and imaging time
The voxel size of a 2D MRI image is defined by the slice
thickness, and the in-plane resolution Dx and Dy (see
Data S1). Note that MRI is not limited to 2D imaging, as a
genuine three-dimensional (3D) dataset can be acquired by
applying an additional phase encoding (NPE2) in the slice
selection direction. The imaging time Texp of a standard 3D
MRI experiment is given by the product of NA (the number
of times the experiment is repeated/averaged), NPE1 and
NPE2 (the number of phase-encoding steps in two directions), and TR (the repetition time). Doubling NA does not
double the SNR of the image; rather it increases it by a
factor of 2 as the signal itself increases by a factor of two
and the noise by 2. As the noise (called Johnson noise) is
affected both by the temperature and the resistance of the
RF resonator and the sample, the cooling of the RF resonator can be used to improve the SNR. Cryoprobes which
cool the RF resonator but do not alter the temperature of
the sensitive samples are commercially available, and have
shown promising results in in vivo pre-clinical MRI (http://
www.bruker-biospin.com/mricp-applications.html). As mentioned earlier, the signal from a voxel of a standard MRI
experiment depends mainly on the spin density and the
timing of the pulse sequence used. If the imaging parameters stay the same and the resolution is doubled in each
dimension, the number of spins within a voxel will be
reduced by a factor of eight. To achieve the same SNR as in
the lower-resolution image the whole experiment has to be
repeated 64 (82) times. Hence, experiments that took 1 h will
take now almost 3 days, which is unacceptable for most
experiments. To reduce scanning time, several rapid imaging techniques have been developed. A detailed description
is given in Data S2.
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THE TRADE-OFF BETWEEN PHYSICS AND PLANT
PHYSIOLOGY
Magnetic resonance images which are informative in relation to structural and/or functional features can be created
by various means (Kockenberger et al., 2004; Kockenberger
and Granwehr, 2009; Van As et al., 2009). For most biologists, the complexities of the MRI methodology and NMR
hardware may be hard to comprehend, while their biological
background generally prevents them from making informed
experimental decisions to fully exploit the potential of MRI
technology. Standardized protocols, inasmuch as they exist
at all, are geared to clinical practice. While the physicist aims
to optimize the methodology by considering what combination of MR approaches is most likely to maximize the
information content of the image and spectroscopic data,
the physiologists requirements also need to be considered.
The successful introduction of MRI into plant science
therefore demands a close collaboration across two disciplines which are rather unfamiliar to one another.
The plant biologist used to seeing high-resolution opticalbased images naturally expects the quality of MRI images to
be at least as good. Such images are best generated when
the water molecule is targeted, because its hydrogen nuclei
provide a strong source of magnetization. Examples of fineresolution 1H-MRI images obtained from plant material by
this means include the spiral-shaped array of chloroplasts
present inside of the cells of the alga Spirogyra (Ciobanu
et al., 2002; Ciobanu and Pennington, 2004), for which a
resolution of 3.7 3.3 3.3 lm3 was achieved. A second
example relates to the geranium petiole, resolved to
2 2 50lm3 (Lee et al., 2001). Theoretically, using MRI
(Glover and Mansfield, 2002) a resolution of approximately
1 lm could be reached, but that has not been, to the best of
our knowledge, realized as of today.
Optimizing spatial resolution is a major objective, which
implies the use of high magnetic and gradient field strengths
(Lee et al., 2001; Ciobanu et al., 2002). The strongest currently commercially available MRI microscope has a magnetic field strength of 20 T (this corresponds to a 1H resonant
frequency of 850 MHz, which is almost 14-fold stronger than
a conventional clinical MRI scanner; see Figure S1). The
trade-off is the orientation and the bore diameter of the
instrument. Most plants are highly sensitive to the direction
of gravity, so the orientation of the main magnetic field needs
to be vertical (Chudek and Hunter, 1997). While a human
system usually has a horizontal bore with a diameter of
60 cm, the diameter of a vertical bore of high field NMR
systems (e.g. 20T) is 10 times smaller. Thus, the use of such a
system is restricted to small specimens. The typical size of a
wide bore high-field system [the two leading manufacturers
are Bruker Biospin (http://www.bruker-biospin.com/) and
Agilent Technologies (http://www.agilent.com/)] is bigger
and reaches 8.9 cm in diameter. Plants of moderate size, such
as Arabidopsis thaliana or part of plants (e.g. pods, leaves or

seeds) are small enough to be subjected to high-field MRI.
Despite imaging whole plants, specific regions can be
targeted by the judicious placement of the RF resonators, a
measure which simultaneously enhances sensitivity and
resolution, and shortens the measurement time. The construction of these RF resonators can require technical innovation (Neuberger and Webb, 2009).
In some respects, plants are a more convenient experimental subject than are humans. A major advantage lies in
the possibility of performing signal averaging over many
hours, which is obviously inappropriate in the clinical setting
where the length of time a patient can remain still is limited.
This problem does not arise in plant specimens, which
appear to be less sensitive to both the noise and strength of
the magnetic field associated with MRI (Osuga and Tatsuoka,
1999; Paul et al., 2006), thus allowing both long-duration
experiments and a wide range of short and intensive
excitation pulses to be applied (Blumler et al., 2009); as a
result, experiments can be focused on very high resolutions
and/or quantifying the dynamic behaviour of tissues.
Further problems arise when long-term experiments are
conducted as the plants need to be fully supplied with water,
nutrients and light. To ensure this, customized controlled
climate chambers have been devised (Van As, 2007). However, not much space is available for the insertion of a
climate control device into the NMR system. A clever
approach is the use of split coil magnets which provide
opportunities to work with larger objects (Figure S1). While
very high-resolution images are not available due to the low
field strength, functional imaging with reasonable resolution delivers important results. Anyway, one has to consider
that a high spatial resolution is not always needed, as is
elaborated here and elsewhere (Kockenberger, 2001; Van As
et al., 2009).
MAGNETIC RESONANCE IMAGING FOR OUTDOOR
EXPERIMENTS
To apply MRI to plants growing in their natural environment,
the NMR device needs to be transportable (Van As et al.,
1994; Rokitta et al., 2000; Haishi et al., 2001; Wright et al.,
2002; Goodson, 2006; Blumich et al., 2008). The NMRMOUSE (mobile universal surface explorer) is an example of
such a device. It is small and when placed on the surface of a
specimen it allows the detection of the NMR signal from any
relevant part of a plant within a few centimetres of its surface. Only the region covered by the magnet is investigated
in more detail by the RF resonator. An application of such an
instrument was demonstrated for monitoring leaf water
dynamics spectroscopically (Capitani et al., 2009). The
C-shaped magnet generates a very homogeneous magnetic field (Rokitta et al., 2000; Wright et al., 2002; Utsuzawa
et al., 2005) which is strong enough for imaging purposes.
The suitability of this type of device for anatomical imaging
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has recently been demonstrated by in vivo monitoring of
trees (Kimura et al., 2011; Umebayashi et al., 2011). Currently, however, such devices are relatively heavy to handle
and/or their shape is insufficiently flexible (Halbach, 1980).
A rather elegant solution to this problem is represented by
cut-open force free NMR (NMR-CUFF; Figure S1), in which
the magnet can be readily clamped (and later removed)
around a tree trunk, a branch, a fruit or a plant stem (Raich
and Blumler, 2004; Windt et al., 2011). The prototype device
weighed just 3.1 kg, and was able to provide a flux density of
0.57 T over a 5-mm diameter sphere. The level of resolution
obtained by NMR-CUFF remains limited, but this restriction
is likely to be lifted by ongoing technical improvements in
magnet design, which have seen the field strength and
homogeneity generated by NMR-CUFF reach comparable
levels typical for clinical fixed MRI device supplied 20 years
ago.
IMAGING OF PLANT DEVELOPMENT
A major thrust of developmental biology is to understand
how molecular and cellular processes produce 3D morphology. With its non-invasive character MRI is, unlike other
imaging techniques, capable of gaining information with
high spatial resolution, both structural and biochemical, as
well as on temporal changes within the plant, and can
therefore be used to monitor plant development processes.
Seed and bulb germination
One of the fundamental plant processes particularly amenable to MRI analysis is germination, which begins with the
uptake of water into the seed (imbibition). As MRI does not
require tissue transparency for image acquisition, it can be
used to non-invasively trace the fate of imbibed water in
seeds, and thereby identify which tissues are involved in
water distribution. The germination process has been studied by MRI in most leading crop species (wheat: Rathjen
et al., 2009; maize: Ruan and Litchfeld, 1992; legumes:
Wojtyla et al., 2006; Garnczarska et al., 2007a; barley: MolinaCano et al., 2002) and other species including trees
(Kockenberger et al., 2004; Roh et al., 2004; Terskikh et al.,
2005; Kikuchi et al., 2006). Tobacco seeds are as small as
1 mm in diameter, and could be imaged in vivo during their
imbibition and germination by Manz et al. (2005) at a level of
resolution sufficiently high to visualize the tissue-specific
water penetration pathway and to characterize the dynamics
of water uptake. In beans, an unexpected mechanical vibration of the seed was observed during the imbibition process
(Kikuchi et al., 2006), and some of the regulatory mechanisms controlling the uptake of water were revealed
(Koizumi et al., 2008). A future task will be the integration of
in vivo MRI data with those on the complex gene regulatory
and metabolic networks controlling seed germination
(Bentsink et al., 2010). Germination is an important crop trait
and application of non-invasive techniques, especially such
as MRI, have the potential to facilitate crop improvement by

contributing to both experimental and agricultural practice
(e.g. evaluation of seed composition, quality, screening
procedures and others). Monitoring water uptake has relevance for the food industry/biotechnology. As a result of the
information gained from the NMR images, maltsters can
improve the efficiency of the malting process (Horigane
et al., 2006).
Some plant species have evolved the capacity to form
storage bulbs as a vehicle for vegetative propagation, and
their formation can be recognized quite early by the onset of
certain changes in the internal structure of the relevant part
of the plant. The non-invasiveness of MRI allows for the
visualization of these changes in a way that is impossible to
achieve non-destructively using conventional microscopy
(Faust et al., 1997; Ishida et al., 2000; Ratcliffe et al., 2001).
The higher free water content of actively developing organs
within the bulb (inflorescence, florets, leaves) will result in a
hyperintense signal in the MRI images (Van der Toorn et al.,
2000). Structural, physiological and metabolic changes
taking place inside the bulb can also be monitored (Robinson et al., 2000; Van der Toorn et al., 2000; Roh et al., 2004).
Magnetic resonance imaging of Lachenalia aloides bulbs
revealed the effect of elevated temperature on various
internal storage processes (Roh, 2005). Bud development
and dormancy induction in woody plants is also associated
with water content and mobility. Magnetic resonance
imaging can be used to examining the behaviour of water
in buds and contribute to investigations on the mechanisms
underlying the adaptation of plants to environmental and
climate changes (Tanino et al., 2010; Kalcsits et al., 2009;
Yooyongwech et al., 2008).
Seed development
Developing seeds are valuable targets for MRI, and various
approaches have been used to characterize this phase of the
plant life cycle. Glidewell (2006) used MRI to study developing barley grains from anthesis to maturity, generating 3D
images of caryopses as well as quantitative T2 maps (see
Data S1). Chemical shift imaging (CSI; see Data S1) was
applied to detect changes in the tissue distribution of water,
soluble carbohydrates and lipids. Further developments of
the method elucidated quantitative lipid maps (Neuberger
et al., 2008). Gruwel et al. (2008) applied diffusion-weighted
MRI on wheat grain and endosperm pore size. Furthermore,
the embryo cell dimensions could be obtained. Ishimaru
et al. (2009) used MRI to explain the formation of distinct
phenotypes of rice grains grown under different temperature
conditions. MRI was combined with physiological measurements, laser microdissection and expression analysis.
Garnczarska et al. (2007b) used MRI to study water content/
distribution during maturation of lupin seeds, elucidating
the spatial/temporal relationship to dehydrin proteins. Melkus et al. (2009) modelled the 3D structure of developing pea
2012 The Authors

(a)
(d)
(b)
(c)
distribution. Windt et al. (2009) were able to demonstrate

that most of the water translocated into the tomato fruit
travels through the xylem and not the phloem, thereby
resolving a long-standing difficulty in modelling fruit
growth. Magnetic resonance imaging has also found applications in the study of certain parameters of fruit quality
(Chudek and Hunter, 1997; Musse et al., 2009; Haishi et al.,
2011).
Root growth
(e)
Most recent improvements in MRI technology have enabled

the investigation of root development. Magnetic resonance
imaging can visualize the 3D geometry of roots not only in
liquid or clear media but also inside soil or sand (Kaufmann
et al., 2009; Blossfeld et al., 2011; Hillnhutter et al., 2011).
This allows access to the hidden root architecture and how
it relates to local soil composition, environmental and biotic
factors.
IMAGING OF WATER DYNAMICS IN LIVING PLANTS
Figure 2. Non-invasive study of seed structure and metabolite distribution in

pea during early developmental stages.
(a) Hand section through the pod and seeds showing seeds filled with liquid
endosperm.
(b) Three-dimensional NMR-based model of pea seed.
(c) Selected longitudinal section from NMR three-dimensional dataset used
for modelling.
(d, e) Distribution of sucrose (in d) within the embryo sac showing elevated
levels in endosperm versus suspensor and gradient distribution of sucrose in
the embryo; sucrose concentration is colour-coded. Image in (e) represents
the reference image (cross-section) showing the seed coat, embryo, endosepermal vacuole and suspensor. For more information, see Melkus et al.
(2009).
Abbreviations: e, embryo; ev, endospermal vacuole; sc, seed coat; s,
suspensor.
seeds, quantifying the volume ratio of different seed organs

including the tiny suspensor. Magnetic resonance imaging
was linked to NMR spectroscopy and allowed quantification
of local concentrations of metabolites in different regions of
the seed (Figure 2, Video clip S1). Hayden et al. (2011)
applied an MRI approach for the integrative study of seed
development in two oat cultivars, combining lipid mapping,
metabolite and transcript profiling.
Fruit growth
Glidewell et al. (1999) monitored the whole developmental
process of blackcurrant (Ribes nigrum) fruits attached to the
plant. Their use of various gradient echo imaging
sequences, chemical shift effects, etc. and both 2D and 3D
reconstructions allowed for a correlation between NMR
signal intensities and specific tissue features, such as cell
size, air inclusions and lipid content. The quantification of
fruit composition in oil palm carried out by Shaarani et al.
(2010) identified a tissue-specific pattern of oil and water
The distribution of water, nutrients and regulatory compounds in plants relies on the functions of the vascular
system. This system, consisting of phloem and xylem, is
deeply embedded in plant tissues, thus any functional
investigation becomes a technical challenge. To explain the
driving forces of solute movement within the vascular system two theories have been proposed: the cohesiontension
(CT) theory (Dixon and Joly, 1894) to explain xylem transport
and the pressure-flow hypothesis (Munch, 1930) to explain
phloem transport. Their validity has remained a matter of
debate over decades.
The development of non-invasive NMR based technologies created a basis for in vivo study of xylem and phloem
transport in living plants (Kockenberger, 2001). Pioneering
flow MRI by Kockenberger et al. (1997) was followed by the
development of fast imaging techniques such as fast lowangle shot (FLASH; Rokitta et al., 1999; Peuke et al., 2001)
and q-space imaging (Scheenen et al., 2007). Dedicated
hardware was developed and strategies to visualize and
quantify dynamics of the plant vascular system in wide
ranges of plant species were proposed (Windt et al., 2006;
Van As, 2007; Van As et al., 2009). Currently, xylem and
phloem flow and their mutual interactions is one of the most
popular subjects for MRI (Holtta et al., 2006; Van As, 2007;
Windt et al., 2009). A particular contribution of MRI has been
the in vivo characterization of fluxes in xylem/phloem
(Figure 3), the quantification of the diurnal pattern of solute
flow, monitoring of embolism repair and the defining of
certain structurefunction relationships, such as between
sieve tube geometry and phloem flow (Peuke et al., 2001;
Salleo et al., 2004; Kaufmann et al., 2009; Mullendore et al.,
2010). Examples of important in vivo observations include
the facts that water flow through the plant as a whole
responds both to the nitrogen source and root/stem cooling
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(a)
(c)
(b)
(d)
Figure 3. High resolution magnetic resonance imaging (MRI) demonstrating

water flow dynamics in tomato.
(a) Tomato truss; arrow shows the peduncle connecting tomato fruits to the
stem.
(b) Microscopy image (light microscopy) of the perimedullary tissue showing
localization of phloem (ph) and xylem (x).
(c) Volume flow map of influx and efflux in the peduncle before truss pruning.
Influx in the outer ring is shown in blue and efflux in red. Influx in the inner
ring is shown in green. The influx in the inner ring corresponds with the
position of the perimedullary tissue.
(d) High-resolution colour-coded quantitative volume flow map (see colour
bars on the top panel). Image courtesy of Dr C. Windt, Forschungszentrum
Julich GmbH, Germany. For more information, see Windt et al. (2009).
(Scheenen et al., 2001; Peuke et al., 2006, Schulze-Till et al.,

2009). Takase et al. (2011) exploited MRI to relate the solute
flow to the expression of aquaporin genes in A. thaliana. The
growing consensus is that the plant vascular network, far
from being a passive plumbing net, is in fact a finely
regulated transport system.
FUNCTIONAL IMAGING OF THE ABIOTIC STRESS
RESPONSE
The geographical dispersion of a plant species is known to
be greatly affected by the frequency with which abiotic
stresses, in particular drought, salinity, cold or heat, are
experienced (Boyer, 1982; Araus et al., 2002). The common
link between all these stresses is that at least some of their
detrimental effect is caused by a disruption to the plants
moisture status (Verslues et al., 2006).
Drought stress
Magnetic resonance imaging was attempted in 1986 on
intact drought-stressed Vicia faba plants (Bottomley et al.,
1986) and Pelargonium spp. (Brown et al., 1986), but it has
only recently become possible to revisit these pioneering

experiments using current MRI equipment and methodology (Van der Weerd, 2002a; Scheenen et al., 2007). While
conventional physiological experiments have tended to
focus on the response of particular organs, 1H-MRI is well
suited to the study of stress responses more holistically. In a
MRI-based comparison between maize and pearl millet
(Pennisetum glaucum), Van der Weerd et al. (2001)
demonstrated differences in the drought response of the two
species.
The stem is the logical location for an MRI probe, because
it connects the root with the leaf, and its tissue architecture is
highly suited to the acquisition of high-resolution images
(Van der Weerd et al., 2002b). Xylem embolism (where
solute flow is blocked by an air inclusion) is one of the early
effects of drought stress. Various hypotheses have been
proposed to explain how such embolisms can be corrected
(e.g. Salleo et al., 2004), but so far none has been fully
validated. Magnetic resonance imaging provided the first
direct observations of xylem cavitation and embolism repair
in an intact plant (Holbrook et al., 2001), experiments which
were later extended to a wide range of species (Clearwater
and Clark, 2003; Scheenen et al., 2007; Kaufmann et al.,
2009).
The root is increasingly recognized as a key player in the
adaptation of plants to drought stress (Pennisi, 2008; Lopes
et al., 2011). Magnetic resonance imaging can generate
images of roots in soil, modelling their structure, monitoring
moisture changes in the rhizosphere and carrying out
functional studies of plant nutrition (Pohlmeier et al., 2008;
Blossfeld et al., 2011; Hillnhutter et al., 2011). Spin density
MRI analyses of drought-stressed maize roots have successfully localized cavitation events and allowed the visualization of the refilling process, shedding light on the identity
of certain in vivo processes underlying drought tolerance
(Kaufmann et al., 2009).
The response of plants to field drought is more complex
then that induced in controlled experiments, in which care is
taken to ensure that drought is the sole stress being
imposed. Magnetic resonance imaging experiments carried
out in the field allow us to monitor integrated plant
responses instantly at the time and place they arise (Capitani
et al., 2009; Windt et al., 2011). Quercus ilex leaves have
been used to monitor what changes occur in vivo over the
course of progressive drought (Sardans et al., 2010). Here,
measurement of the water content in the plate and reap of
the leaf indicated a non-homogeneous response to stress.
This information suggests how gene expression studies
could be based on topographical information. Progress in
this direction may deliver the use of MRI as a diagnostic tool
to help the scheduling of irrigation. It could also be
developed into a crop breeders selection tool for identifying
genetically superior individuals with respect to water use
efficiency.
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(a)
(c)
(b)
(e)
(d)
Figure 4. Comparison of light and magnetic resonance (MR) microscopic imaging for the study of rehydration in the African resurrection plant Myrothamnus
flabellifolia.
(a) The plant before and after watering, demonstrating the rehydration potential.
(b) Three-dimensional reconstruction measured on a dry intact Myrothamnus branch at a height of 5 cm.
(c) Light microscopy image of a cross-section of an air-dry Myrothamnus branch stained with the lipophilic dye Nile red. Yellow fluorescence indicates lipids (p, pith;
lta, lipid-rich tracheid assemblies; lt, leaf trace).
(d) High-resolution 1H-NMR lipid distribution images of an air-dry Myrothamnus branch (pp, pith periphery; lp, lipid pieces).
(e) 1H-NMR imaging visualizes non-invasively the spreading of water (bright areas) within a virtual cross-section of an air-dry branch during rehydration. For further
details see Schneider et al. (2003).
Some plant species have evolved a very high level of

drought resistance (Schneider et al., 2003; Liu et al., 2007),
and MRI might help to unravel the underlying mechanisms.
The African resurrection plant, Myrothamnus flabellifolia, is
able to switch between a highly desiccated state and a fully
hydrated green plant within 24 h of watering (Figure 4).
Lipid composition, water movement within its shoots and
leaves during drying and rehydration episodes have been
visualized by Schneider et al. (2003) using MRI. This analysis
provided evidence that the key transport tissues are
equipped with lipids, and that the spatial arrangement of
the xylem enables repeated cycles of hydration and dehydration in an organized manner.
Cold stress
Episodes of low-temperature stress challenge plants in a
multitude of ways, and the responses should be considered
as a syndrome rather than as a single reaction (Beck et al.,
2007).
Prolonged exposure to near freezing temperatures can
cause functional changes and tissue damage. Continuous
MRI-based monitoring over a 240-h period allowed a
detailed study to be made of the in vivo response to cold
stress of woody lianas (Clearwater and Clark, 2003). Scheenen et al. (2002) used a combination of the imaging of flow
and T2 to analyse the effect of cooling the roots on the water

status of intact cucumber plants. Their major findings were
that cooling induced a substantial decrease in water uptake,
due to a root response and embolisms in the xylem
(Scheenen et al., 2007). These authors also reported the
restoration of functioning xylem, as observed by flow, not
only filling of vessels. Cooling the stem of Ricinus communis
caused both the leaching of sucrose from the stem phloem
vessels and the short-term inhibition of mass flow at the
beginning of cold treatment (Peuke et al., 2006).
An early consequence of cold stress caused by exposure
to sub-zero temperatures is the dehydration of tissues due to
the freezing of water, and the subsequent damage to
membranes upon thawing (Yamazaki et al., 2008; Yadav,
2009). 1H-MRI is well suited to detect how the plant cell
copes with the nucleation and expansion of ice (Ishikawa
et al., 1997; Ide et al., 1998), because it can monitor the
development of hardening and de-hardening, which is
difficult to achieve using a destructive assay. It also allows
for monitoring over prolonged periods of exposure in the
natural environment. As long ago as 1995, MRI was used to
investigate freezing tolerance in wheat (Millard et al., 1995).
The MRI analysis was able to identify clear behavioural
differences between acclimated and non-acclimated plants.
It was even possible to identify the most cold-sensitive part
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of the plant. Non-invasive approaches are also advantageous for forest species. The flower buds of hibernating
trees differ in their susceptibility to freezing damage. Methods have been developed based on MRI to determine the
response to sub-zero temperatures of various tree species
(Ishikawa et al., 1997; Price et al., 1997; Ide et al., 1998).
Comparisons of MRI images obtained from trees exposed to
freezing conditions have revealed that Acer japonicum
flower buds, leaves and stem bark tissue had become frozen
when the temperature fell to )7C, but the lateral primordia
retained their viability down to )40C. This strategy (harmonized freezing) might help to ensure the survival of trees. In
Arabidopsis, freezing tolerance is associated with the avoidance of damage to the plasma membrane and/or membrane
repair (Yamazaki et al., 2008). A better understanding of the
mechanisms underlying freezing tolerance at the whole
plant level will require a determination of the relationships
between ice management, cell wall properties and membrane resealing, and this may be more easily achieved by
incorporating MRI analysis with more conventional biological approaches.
THE HOSTPATHOGEN INTERACTION
Plants are challenged by a range of viral, bacterial and fungal infections, and an intimate and dynamic means of
monitoring the hostpathogen interaction would greatly
enhance our understanding of the infection process.
Although the damage caused by infection is often readily
visible, it can sometimes remain hidden within the plant.
A good example of the use of MRI in a plant pathology
context has been given by an analysis of diseased sycamore
(Acer pseudoplatanus) trees, in which both the various
infection pathways exploited by diverse pathogens could be
well defined and the effect of disease on the water status of
the wood monitored (Pearce et al., 1994). MacFall et al.
(1994) imaged gall formation in pine seedlings following
their infection by the bacterium Chronartium quercuum.
A further example is described by Goodman et al. (1996)
regarding the fungus Botrytis cinerea, where 3D MRI datasets were used to successfully define the location of infected
regions inside diseased strawberry fruit. It appeared that the
pathogen breaks down parenchymal cell walls, with the
result that cell contents leaked into the intercellular spaces
(Goodman et al., 1996; Chudek and Hunter, 1997). The
application of MRI contributed to the understanding of
Pierces disease as well, which was a major problem in some
Californian vineyards during the early 1990s. It had been
assumed that the sole pathogen involved was the bacterium
Xylella fastidiosa which colonized the xylem, and thus
compromised water translocation throughout the plant.
However, MRI analysis showed that the blockage of the
xylem resulted from the hosts active responses to infection
rather than from the proliferation of the pathogen itself
(Alonso et al., 2007).
Jatropha curcas, a potential source of biodiesel, is generally regarded as being a hardy, drought- and diseaseresistant plant, but it can be heavily damaged by the
whitefly-borne Jatropha mosaic virus. Sidhu et al. (2010)
have recently demonstrated the value of MRI and highresolution magic angle spinning NMR spectroscopy for
studying this viral infection. The contrast of T1 and T2
weighted images detected differences in the spatial distribution of water, lipids and macromolecules in infected
versus healthy stems. Alterations in certain anatomical
structures and in the rate of sap translocation could then
be correlated with metabolic changes in infected plants.
Pine wilt disease is characterized by the formation of
embolized tracheids following the invasion into the resin
canal of the pine wood nematode Bursaphelenchus xylophilus. Infected trees eventually die as a result of compromised xylem conductivity. Umebayashi et al. (2011) devised
a compact MRI system featuring a C-shaped magnet and a
movable U-shaped RF coil, which allowed the trunks
internal structure to be imaged at a high level of resolution.
The dynamics of disease spread and the resulting damage
could then be precisely documented. These experiments
suggest a future place for MRI-based devices in the early
diagnosis of some tree diseases.
Current developments in MRI have allowed the noninvasive detection of below-ground symptoms in sugar beet
caused by the beet cyst nematode and/or soil-borne root rot.
Magnetic resonance imaging monitored a synergistic relationship between the two pathogens, providing new insight
into plantpathogens interactions (Hillnhutter et al., 2011).
SUSTAINING BIODIVERSITY
Biodiversity is threatened by a combination of over-exploitation, pollution and climate change, raising the priority of
conserving plant genetic resources. Seed storage under low
temperatures represents an efficient means of preserving
many flowering plant species, while some non-seed tissues
(e.g. tubers, bulbs, meristems) can be cryopreserved. The
longevity of seeds is an issue in all germplasm banks (Nagel
and Borner, 2010), and the lack of non-destructive methods
to assess seed viability means that seed numbers inevitably
become depleted over time, forcing stocks to require
regeneration on a regular basis. The ability of MRI to provide a non-invasive assessment of the integrity of a seeds
internal structure, to detect the presence of internal pathogens (Kockenberger et al., 2004), to visualize the distribution
of lipids or water within a seed (Ishida et al., 2004; Neuberger et al., 2008) and to monitor the physical state of
moisture within a seed as a result of storage at low temperatures (Borompichaichartkul et al., 2005) are all highly
relevant for developing an efficient germplasm conservation strategy. Systematic comparisons of the structure and
composition of freshly harvested versus stored seeds
(possibly augmented by artificial seed ageing measures)
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could succeed in defining what parameters are associated
with seed viability (Gruwel et al., 2002; Borisjuk et al., 2011).
Scaling MRI techniques appropriately and developing a
cost-effective hardware platform will be needed to promote
the application of MRI in this area. It should be noted that
electron paramagnetic resonance (EPR) in combination with
the use of spin probes offers an alternative mean for
non-invasive observation of seed viability and longevity
(Golovina et al., 2010).
Most parts of the plant cannot be maintained intact over
the long term, and are substantially altered during fixation or
cryopreservation procedures. The creation of a virtual library
providing 3D models of these materials based on MRI data
of living plants could enable a indispensable digital
collection of a mass of biodiversity information and make
it accessible for future generations of scientists. Efforts are
under way to develop appropriate hardware, software and
methodology.
GENE EXPRESSION AND FUNCTION
Prospects for employing MRI reporter genes
Currently exploited reporter genes, such as those encoding
b-glucuronidase, luciferase or GFP, are based on histochemical staining or fluorescence. Optical projection
tomography has extended the resolution of these reporters
in plant material to three dimensions within a single cell, or
in some cases within tissue sections with a thickness up to
15 mm (Lee et al., 2006; Truernit et al., 2008). The current
peak resolution achieved in animal material is represented
by the transgenic brainbow mouse, in which the simultaneous expression of multiple fluorescent proteins has
resulted in the recognition of some 90 distinguishable colours (Livet et al., 2007). Two new promising classes of
reporter genes are now emerging, one of which relies on
affinity for specific radioisotopes (Serganova et al., 2007)
and the other on MRI (Gilad et al., 2008). A particular feature
of MRI reporter genes is that in principle they can combine
gene expression data with anatomical and functional information. In the most advanced of these, the reporter gene
product interacts with a reagent containing the element
gadolinium (Gd) (Gilad et al., 2008). The Gd enters the root
cell symplast, moves in conjunction with the flow of solutes
and can be well traced in plants (Gussoni et al., 2001; Zhang
et al., 2009). Gadolinium is non-toxic for plants, both in its
chelated and unchelated forms (Quiquampoix et al., 1990),
but its membrane permeability needs to be considered.
Another opportunity is provided by the Escherichia coli gene
encoding polyphosphate kinase (PPK) (Ki et al., 2007).
Polyphosphate kinase does not require an exogenously
supplied substrate and can be visualized by 31P-MRI. The
enzyme catalyses the synthesis of inorganic (largely immobile) polyphosphate from ATP, and has been expressed
constitutively in plants (Van Voorthuysen et al., 2000;
Nagata et al., 2006). A disadvantage of this system is the low

sensitivity of 31P-NMR. A further option is the use of ironbased reporter genes (Hill et al., 2011), which are associated
with good contrast in 1H-MRI. The heterologous expression
of ferritin genes has been achieved in a number of plant
species, but aspects related to the accumulation of iron and
its complex regulation complicate the picture (Van Wuytswinkel et al., 1999; Drakakaki et al., 2000; Jiang et al., 2006).
Finally, the switchable chemical exchange saturation transfer (CEST)-based reporter genes (Liu et al., 2011) have the
feature that they are able to simultaneously visualize more
than one target. As yet MRI reporter genes have not been
developed for plant material, but it is likely that they will be
in the future.
Bridging the gap between gene expression and function
The non-invasive monitoring of plant processes in vivo
offers the potential to establish relationships between gene
expression and physiological events, which can help in the
elucidation of gene function. The role of aquaporins in
maintaining plant moisture status, water hydraulics and
stress tolerance has been controversial for some time
(Katsuhara et al., 2008), but their visualization using 1H-MRI
has resolved much of the argument. When Takase et al.
(2011) monitored the behaviour of water in the A. thaliana
root, a diurnal pattern of water content was observed in the
basal zone of the root, and this rhythm was maintained even
when the plants were kept under continuous light or darkness. Imaging data were compared with the expression
profiles of two aquaporin-encoding genes, known to control
water uptake (Chaumon et al., 2005) and whose expression
followed a circadian rhythm under continuous light. The
circadian oscillation in water dynamics was abolished in a
mutant compromised for the detection of the circadian signal (Liu et al., 2001). Thus the inclusion of MRI data allowed
a linkage between function (water dynamics) and gene
expression. The wider use of MRI for this sort of research can
be expected to yield many novel insights into gene function
(Yooyongwech et al., 2008).
Another example is the Jekyll-gene in barley which has
been shown to have a role in sexual reproduction (Radchuk
et al., 2006). The localized up-regulation of Jekyll appears to
be coupled with cell autolysis in the developing grain, while
its down-regulation slows the growth of the endosperm. On
this basis, it was suggested that the function of JEKYLL
is associated with the allocation of nutrients between
maternal (pericarp) and filial (endosperm) tissue. Later,
13 1
C/ H-MRI was applied to visualize allocation of 13C sucrose
in plants engineered to repress Jekyll expression to various
extents (Melkus et al., 2011). These experiments showed
that the quantity and distribution of sucrose were dependent on the degree of Jekyll repression, approving the role
of JEKYLL in nutrient allocation during the process of grain
filling.
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The analysis of mutant plants
Mutants have proven invaluable for defining gene function,
but not uncommonly their primary effect is concealed by
pleiotropy. In such cases, non-destructive methods may be
required to identify the primary effect of the mutation, presenting an opportunity for MRI, based on its ability to
simultaneously monitor a range of structural, metabolic and
physiological parameters. This type of analysis is rare in the
plant world as yet, and MRI technology is still challenging
when applied to small targets such as seeds of A. thaliana.
Fortunately, the novel model plant species (rapeseed, rice,
maize, etc.) should be more amenable to MRI.
Fast Seefeldt et al. (2007) were able to use 1H-NMR
imaging to both identify and characterize b-glucan (BG)
mutants in barley. The presence in food of BG lowers both
its cholesterol content and glycaemic index. Magnetic
resonance imaging was proved to be effective for delineating the internal structure of the grain, and for identifying
varietal differences in the grains water-holding capacity.
Another use of MRI was to characterize a pea mutant
(Borisjuk et al., 2002), as part of a wider attempt to understand the role of the liquid endosperm. Applied to the seed
of a mutant which develops a giant endosperm, MRI was
able to determine non-destructively 3D structures and the
volume of each of the seeds component organs (Melkus
et al., 2009). Both the concentration and the distribution
inside the liquid endosperm of some major metabolites
were obtained in vivo. The endosperm is the major seed
storage organ in monocot crop species, and NMR spectroscopy has been widely applied to help understand the
Figure 5. Quantitative imaging of lipid in a living

barley grain. (a) Fragment of a barley spike used
for the magnetic resonance imaging (MRI) analysis. (b) Longitudinal tissue section showing the
internal structure of the grain. (c) Lipid staining in
a longitudinal tissue section using Sudan/ethanol procedure (lipids stained in red). (d) An MRI
based three-dimensional model of the spike
shown in (A) (see also Video clips S2 and S3 (e)
Non invasive visualization of the spike demonstrating the internal structure of grains/spike with
resolution of 35 lm. (f) Quantitative map representing lipid deposition within the grain in vivo;
lipids are mainly found in the embryo and the
aleurone layer; lipid content is colour coded.
Abbreviations: al, aleurone layer; em, embryo;
en, endosperm; np, nucellar projection; p, pericarp. For further details see Neuberger et al.
(2008).
metabolism of the endosperm and its regulation (Alonso

et al., 2011). Linking such efforts with MRI should accelerate
progress in this field.
IMAGING OF PLANT METABOLISM
The study of plant metabolism and its compartmentalization
provides a number of potential MRI applications, since the
technology offers the non-invasive measurement of various
metabolite concentrations (Bourgeois et al., 1991; Soher
et al., 1996; Vanhamme et al., 1997; Tkac et al., 1999; De
Graaf, 2007). In order to be informative for the biologist, MRI
data have to be related to known histological, biochemical
and other characteristics of the tissue, and this represents an
area where substantial progress has been achieved in recent
years in particular, in the imaging of the commonest
assimilates exported into and distributed within the developing seed, and in the quantification of seed storage compounds.
Visualization of lipid storage and degradation
Regulation of oil storage activity in vivo is complex and
requires non-invasive approaches. Various NMR-based
methods have been used for lipid detection both in dry plant
material and in oil-rich fruits/seeds (reviewed in Neuberger
et al., 2008). When CSI was employed as a non-invasive
means of visualizing lipid distribution in the mature soybean
seed, clear lipid gradients were observable, in accordance
with the differentiation pattern of the plastids, which are the
site of fatty acid synthesis (Borisjuk et al., 2005). A disadvantage of CSI is its relatively long experiment time. Hence,
it is only of limited use for delivering a reliable picture of
(a)
(b)
(c)
(d)
(e)
(f)
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Figure 6. Quantitative imaging of lipid in a living
oat grain.
(a) Oat grain pictured using a light microscope.
(b) Cross-tissue section showing the endosperm
and pericarp.
(c, d) Quantitative map representing lipid deposition within the grain in vivo (corresponding to
the cross-section shown in (b)) in the low-oil
cultivar Freja (c) and the high-oil cultivar Matilda
(d). Lipid content is color-coded.
Abbreviations: al, aleurone layer; en, endosperm; em, embryo; p, pericarp. For further
details see Hayden et al. (2011).
(a)
(b)
(c)
(d)
events within a developing seed. In a more recently developed approach, reliance was placed on the slightly different
resonance frequencies of water and lipids, which could be
exploited using a frequency-selective MRI technique (Neuberger et al., 2008). This method shortened the measurement time up to 10-fold, and delivered a spatial resolution
close to the cellular level.
The simultaneous imaging of anatomy and lipid deposition offers the opportunity to relate lipid accumulation with
seed development. Using this approach in the developing
barley grain revealed concentrated lipid deposition in particular regions of the embryo (scutellum and nodule), as well
as in the aleurone layer of endosperm, a structure which is
only a few cell layers thick (Figure 5, Video clip S2). At the
same time, the regions where lipid degradation occurs later
in the maturation process were identifiable. In high-oil
cultivars of oat, lipid occupies the entire endosperm as
demonstrated by the MRI-based analysis (Figure 6, Video
clip S2). To date, this mode of lipid mapping has been
applied to seeds of oat (Hayden et al., 2011), oilseed rape
and barley (Neuberger et al., 2009) as well as tobacco, maize,
wheat, Jatropha, pine, cotton, linseed and sacred lotus (our
own unpublished data). Combining oil topology with the
analysis of gene expression and metabolites has the
potential to identify key factors in the regulation of lipid
metabolism in vivo (Hayden et al., 2011) and is expected to
provide novel insights into the control of storage in crops. In
the future one can anticipate an equivalent approach being
taken to study the fate of storage lipids during germination.
Visualization of metabolite distribution
A further focus of MRI relates to the imaging of individual
metabolites, giving information on their distribution, trans-
port and conversion within the cell (Ratcliffe et al., 2001;

Kockenberger et al., 2004). As the particular composition
and architecture of plant tissues reduces the sensitivity of
MRI, only abundant metabolites such as sucrose (Verscht
et al., 1998; Szimtenings et al., 2003) and free amino acids
have been successfully targeted to date. Nevertheless, the
non-invasiveness of MRI has provided a number of analytical opportunities, which are unobtainable by destructive
sampling which induces the wounding response. Chemical
shift imaging has only a minimal requirement for post-processing correction, and the acquisition and processing procedure tends to be relatively simple and robust, because
only a single pulse and phase-encoding gradient are needed
for signal encoding. An example is provided by the use of
1
H-NMR CSI to image metabolite distribution in intact pea
seeds at various stages of their development (Melkus et al.,
2009). Structural FLASH (Haase et al., 1986) multi-slice images were acquired at the end of the CSI protocol in order to
topographically relate the spectroscopic data with the corresponding tissue structures. As a result, it was apparent
that the spatial distribution of sucrose (as well as of glutamine and alanine) within the endosperm vacuole tends to be
rather uniform, but at the same time is notably different from
that in either the suspensor or the cellularized embryo (Figure 2c,d). The sucrose concentration gradient was somewhat different from that of the free amino acids, and was in
accordance with the expression pattern of genes encoding
metabolite transporters. At the same time it was possible to
demonstrate how endosperm metabolite levels respond
both to the onset of storage activity in the embryo and to
specific environmental cues, and to identify the endosperm
glutamine concentration as representing a limiting factor for
protein storage in the legume embryo. Improving the level of
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sensitivity obtainable from small seeds will need some
modification of currently available RF resonators (Neuberger and Webb, 2009). Another MRI application for metabolite
imaging is the study by Wenzler et al. (2008) of carbohydrate
metabolism involved in forming floral nectar (Anigozanthos
flavidus). These authors combined cyclic J cross-polarization and 1H spin-echo imaging (a technique implemented
by Heidenreich et al., 1998) to show the localization of
13
C-labelled glucopyranose and the glucose moiety of
sucrose inside the peduncle during a 13C-feeding experiment.
Dynamic imaging of metabolites
Dynamic NMR protocols (or functional imaging) can be
used for applications beyond the reach of current MRI, such
as attempts to monitor the transport and conversion of
major metabolites. Flow-encoded NMR measurements are
effective where velocities are measured in mm h)1 (Szimtenings et al., 2003; Van As, 2007). However, they are difficult
to perform when velocities lie in the lm h)1 range. The
detection, imaging and quantification of sucrose can be
achieved by using 1H-NMR to target protons associated with
carbon nuclei (Tse et al., 1996; Melkus et al., 2009). The
advantage of using the 1H signal (instead of 13C) is its high
MRI sensitivity. It is impossible, though, to follow certain
sucrose molecules through the plant, and only steady-state
levels are observed. As the natural abundance of 13C is very
low and the dominant 12C isotope is not visible by NMR, a
combination of 13C-NMR and the feeding of 13C-labelled
substrates to the plant can be used to track the 13C-labelled
metabolites on their way through the plant. By combining
NMR spectroscopy and imaging, it is possible to obtain both
metabolic and spatial information regarding 13C-enriched
molecules and their metabolic derivatives from the same
experiment. Various inverse detection schemes have been
developed to further improve the detection sensitivity of the
13
C nucleus (Bax et al., 1983; Rothman et al., 1992). These
pulse sequences provide a range of flexible strategies for the
(a)
(b)
Figure 7. Monitoring of 13C sucrose allocation during onset of seed filling in

barley.
(a) The uptake of 13C in the barley caryopsis occurs by feeding 100 mM 13C
sucrose to the stem (left panel). The red cage shows the position of the
double-resonant 13C/1H-NMR coil.
(b) Visualization of 13C sucrose allocation within the caryopsis (see also Video
clip S3). The time post the start of incubation is indicated. For details see
Melkus et al. (2011).
detection of 13C nuclei and are in general more sensitive than

direct detection of 13C (Heidenreich et al., 1998; De Graaf
et al., 2003).
A recent example of dynamic NMR is given by Melkus
et al. (2011), tracking the allocation of assimilates in barley
seeds. A tool has been developed to not only detect specific
metabolites, but also to produce an adequate level of spatial
and temporal resolution over the course of a prolonged
period of monitoring. In this approach the gradient
enhanced heteronuclear multiple quantum coherence
(geHMQC; Hurd and John, 1991) sequence was applied
using a double-tuned RF resonator and a high magnetic field
strength. The metabolic images were captured either via
direct or inverse 13C detection schemes following 13C feeding.
These results demonstrated for the first time how sucrose
diffuses in vivo inside a developing cereal grain (Figure 7,
Video clip S3). The cellular pathways were identified at a
sub-millimetre level and the tissue-specific velocity of
sucrose allocation was determined. 13C/1H-NMR delivered
a five fold higher in-plane resolution than PET (Jahnke et al.,
2009), and facilitated dynamic observations. Furthermore, in
contrast to MR, PET lacks the information concerning from
which specific molecule a decaying 11C nucleus has originated. The 13C/1H-NMR method allowed for the straightforward co-registration of the structural and the metabolite
images, and therefore the exact identification and localization of metabolites within a tissue. In the barley caryopsis,
the nucellar projection has been shown to represent the
exclusive gateway for sucrose inflow, and possesses several
structural, metabolic and gene expression features enabling
this function (Melkus et al., 2011). Further applications in
other major crops can be expected to identify bottlenecks in
the supply of photo-assimilate to sink organs such as the
seed, thereby providing novel targets for the molecular
(biotechnological) modification of crop species.
Dynamic MRI, metabolic modelling and systems biology
Systems biology is a holistic approach, describing the
complex interactions in biological systems. Magnetic
resonance imaging can substantially contribute to such an
approach because it considers the plants complexity: each
organ comprises distinct cell and tissue types, each of which
may be governed by a distinct metabolic network which all
interact with each other. This compartmentalization needs to
be considered when analyzing the regulation and control of
plant metabolism in vivo (Sweetlove and Ratcliffe, 2011). An
example of the use of MRI for the analysis of metabolic
compartmentalization is represented by an analysis of the
barley endosperm, which was assumed a priori to be metabolically homogeneous. The use of a geHMQC sequence
enabled the detection of 13C alanine, derived by supplying
13
C sucrose to the plant (Rolletschek et al., 2011). Dynamic
imaging was able to demonstrate that 13C alanine synthesis
is restricted to the innermost most hypoxic region of the
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13
(a)
(b)
(c)
(d)
C substrates in order to define the routes by which the

corresponding compounds are taken up, distributed and/or
metabolized. When applying other isotopically labelled nuclei (e.g. 15N, 19F, 31P), one needs to consider the shift in
sensitivity of MRI.
Taken together, we argue that dynamic MRI opens up new
perspectives for the non-invasive analysis of metabolic
compartmentalization, metabolic modelling and the identification of metabolic markers in plants.
ACKNOWLEDGEMENTS
Axel Haase (University of Munich) and Ulrich Wobus (IPK Gatersleben) are gratefully acknowledged for their support in commencing our NMR research on plants. The authors thank Gerd
Melkus (University of California) and Johannes Fuchs (University of
Wurzburg) for dedicated contributions. We also thank Peter M.
Jakob (University of Wurzburg), Andrew Webb (University of
Leiden) and Thomas Altmann (IPK Gatersleben) for continuous
support. We acknowledge funding by the German Federal Ministry
of Education and Research, the Deutsche Forschungsgemeinschaft
and BayerCropScience.
SUPPORTING INFORMATION
(c)
(d)
Figure 8. Functional imaging of metabolite dynamics in the living endosperm

of barley grains by use of magnetic resonance imaging (MRI). (a, b) A MR
image showing the steady-state distribution of 13C sucrose fed to intact
caryopses; the 1H reference image is shown in b. (c) A MR image showing the
localized synthesis of 13C-alanine and its preferential accumulation in the
central endosperm region. (d) Diagram indicating the direction of sucrose
flow (red arrows) within the caryopsis (see also Video clip S3). (e, f) Flux maps
depicting mitochondrial metabolism in peripheral (e) versus central (f)
endosperm regions as derived from flux balance analysis.
Abbreviations: en, endosperm; np, nucellar projection; p, pericarp. For details
see Rolletschek et al. (2011).
endosperm (Figure 8). In combination with biochemical and

flux balance analysis, a spatially resolved metabolic model
of the starchy endosperm has since been derived, with the
aim of obtaining an improved interpretation of localized
metabolic activity. The metabolic compartmentalization
occurring in the starchy endosperm provides a measure of
physiological flexibility, and contributes to the high carbon
conversion efficiency shown by the starchy endosperm of
the cereal (Alonso et al., 2011). Apart from such seed-targeted experiments, a number of related applications are
conceivable. Experimental plants could be fed with various
Additional Supporting Information may be found in the online

version of this article:
Figure S1. Magnetic resonance imaging devices used for microscopic and functional studies.
Data S1. Basic information on magnetic resonance imaging.
Data S2. Overview of rapid imaging techniques.
Video clip S1. Digital model of an individual pea seed, which
permitted a three-dimensional visualization of seed anatomy, and in
particular allowed for the measurement of the volume of various
seed organs.
Video clip S2. Animated three-dimensional model of lipid distribution in mature barley. High lipid signals (in green) are found in the
scutellum and nodule. Lipid in the aleurone is visualized as a blue
layer surrounding the endosperm.
Video clip S3. Animated visualization of sucrose allocation within
the barley caryopsis during grain filling.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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The Plant Journal (2012) 70, 129146

HIGH-RESOLUTION MEASUREMENTS IN PLANT BIOLOGY

Surveying the plants world by magnetic resonance imaging

Received 19 October 2011; revised 6 January 2012; accepted 16 January 2012.

magnetic fields, and the development of new imaging

130 Ljudmilla Borisjuk et al.

Figure 1. Principles of magnetic resonance imaging (MRI).

An MRI system is designed to generate three different

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Surveying the plants world by MRI 131

(Gx,y,z) in a certain manner as shown in the example of a

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as Arabidopsis thaliana or part of plants (e.g. pods, leaves or

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Surveying the plants world by MRI 133

as MRI, have the potential to facilitate crop improvement by

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134 Ljudmilla Borisjuk et al.

distribution. Windt et al. (2009) were able to demonstrate

Most recent improvements in MRI technology have enabled

Figure 2. Non-invasive study of seed structure and metabolite distribution in

seeds, quantifying the volume ratio of different seed organs

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Surveying the plants world by MRI 135

Figure 3. High resolution magnetic resonance imaging (MRI) demonstrating

(Scheenen et al., 2001; Peuke et al., 2006, Schulze-Till et al.,

only recently become possible to revisit these pioneering

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Some plant species have evolved a very high level of

and T2 to analyse the effect of cooling the roots on the water

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Nagata et al., 2006). A disadvantage of this system is the low

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Figure 5. Quantitative imaging of lipid in a living

metabolism of the endosperm and its regulation (Alonso

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port and conversion within the cell (Ratcliffe et al., 2001;

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Figure 7. Monitoring of 13C sucrose allocation during onset of seed filling in

detection of 13C nuclei and are in general more sensitive than

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C substrates in order to define the routes by which the

Figure 8. Functional imaging of metabolite dynamics in the living endosperm

endosperm (Figure 8). In combination with biochemical and

Additional Supporting Information may be found in the online

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Glidewell, S.M. (2006) NMR imaging of developing barley grains. J. Cereal.

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characteristics and dynamics in poplar, castor bean, tomato and tobacco.