NMR Plats

HIGH-RESOLUTION MEASUREMENTS IN PLANT BIOLOGY

Surveying the plants world by magnetic resonance imaging

Ljudmilla Borisjuk1,*, Hardy Rolletschek1 and Thomas Neuberger2,3

1Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrae 3, Gatersleben, Germany,2Department of Bioengineering, Pennsylvania State University, University Park, PA 16802, USA, and3Huck 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 [email protected]).

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 zeugmato-

grams) 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 dedi-

cated MRI facility, along with the rather modest spatial

resolution achieved at the time, optical microscopy main-

tained 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

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 sur-

prisingly 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

2012 The Authors 129The Plant Journal 2012 Blackwell Publishing Ltd

The Plant Journal (2012) 70, 129146 doi: 10.1111/j.1365-313X.2012.04927.x

is possible to generate metabolic maps of the living plant

body, and to use such a data to monitor various physiolo-

gical processes. As an example, an MRI scan of a living

plant stem can demonstrate the location of the xylem ves-

sels, 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

othermethods considered for this type of research are based

on either visible range radiation (confocal laser scanning

microscopy, optical coherence microscopy and optical pro-

jection tomography), on X-rays (high-resolution computed

tomography) or on positrons (positron emission tomogra-

phy, 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/ion-

ization (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 (Hol-

brook 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 Sup-

porting Information (Data S1). Readers who are interested

in more details are referred to a number of excellent text-

books. 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 tech-

niques 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.

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 aMRI 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 non-

zero nuclear magnetic moment (called spin). The most

commonly used in vivo nuclei are 1H, 13C, 19F, 23Na, 31P and39K (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

(a) (b)

(c) (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 theMRI 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.

130 Ljudmilla Borisjuk et al.

2012 The AuthorsThe Plant Journal 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 129146

frequency. The spin magnetic moment of each proton

moves on a randomly oriented cone. The angle between

the spinmagnetic moment and B0 stays constant and the net

magnetization Mz is still zero. Very small fluctuating mag-

netic 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 magnetiza-

tion 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. Thevalues of the nucleus-specific constant c (also referred to asthe gyromagnetic ratio) and the resulting ones of x in amagnetic field of 17.6 T are given in Data S1.

The application of a linear polarized oscillating electro-

magnetic field (B1) with a duration of sB1 at the Larmorfrequency 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 character-

ized 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 rotatedM0 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 trans-

formation. 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 mono-

chromatic, although colour coding can be added. Differ-

ences 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 (seeData 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 direc-

tions), 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 twoand the noise by 2. As the noise (called Johnson noise) isaffected both by the temperature and the resistance of the

RF resonator and the sample, the cooling of the RF reso-

nator 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 men-

tioned 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 parame-

ters 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 imag-

ing techniques have been developed. A detailed description

is given in Data S2.

Surveying the plants world by MRI 131


THE TRADE-OFF BETWEEN PHYSICS AND PLANT

PHYSIOLOGY

Magnetic resonance images which are informative in rela-

tion 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 biolo-

gists, the complexities of the MRI methodology and NMR

hardwaremay be hard to comprehend, while their biological

background generally prevents them frommaking 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 combi-

nation 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 disci-

plines which are rather unfamiliar to one another.

The plant biologist used to seeing high-resolution optical-

based 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 fine-

resolution 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 secondexample 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 ofour knowledge, realized as of today.

Optimizing spatial resolution is a major objective, which

implies the use of highmagnetic and gradient field strengths

(Lee et al., 2001; Ciobanu et al., 2002). The strongest cur-

rently commercially available MRI microscope has a mag-

netic 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 orientationof themainmagnetic 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 leadingmanufacturers

are Bruker Biospin (http://www.bruker-biospin.com/) and

Agilent Technologies (http://www.agilent.com/)] is bigger

and reaches8.9 cm indiameter. Plantsofmoderate 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 con-

struction of these RF resonators can require technical inno-

vation (Neuberger and Webb, 2009).

In some respects, plants are a more convenient experi-

mental 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

themagnetic field associatedwithMRI (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). How-

ever, 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 resolu-

tion 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 applyMRI 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 NMR-

MOUSE (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 sur-

face. 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 mag-

netic 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



has recently been demonstrated by in vivo monitoring of

trees (Kimura et al., 2011; Umebayashi et al., 2011). Cur-

rently, 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, andwas 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 mor-

phology. 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 ame-

nable 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 stud-

ied 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: Molina-

Cano 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 byManz 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 vibra-

tion of the seed was observed during the imbibition process

(Kikuchi et al., 2006), and some of the regulatory mecha-

nisms 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 rele-

vance 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 (Robin-

son 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 devel-

oping barley grains from anthesis tomaturity, 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 mea-

surements, 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. Mel-

kus et al. (2009) modelled the 3D structure of developing pea



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

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 appli-

cations in the study of certain parameters of fruit quality

(Chudek and Hunter, 1997; Musse et al., 2009; Haishi et al.,

2011).

Root growth

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

The distribution of water, nutrients and regulatory com-

pounds 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 sys-

tem 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 technolo-

gies 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 low-

angle 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

(a)

(d) (e)

(b) (c)

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, endo-

sepermal vacuole and suspensor. For more information, see Melkus et al.

(2009).

Abbreviations: e, embryo; ev, endospermal vacuole; sc, seed coat; s,

suspensor.



(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 inA. 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 metho-

dology (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 suc-

cessfully localized cavitation events and allowed the visual-

ization 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.

(a)

(c) (d)

(b)

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 flowmap 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).



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) usingMRI. 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 dehy-

dration 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). Schee-

nen 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 ofRicinus 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

(a)

(c) (d)

(b) (e)

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) Lightmicroscopy 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).



of the plant. Non-invasive approaches are also advanta-

geous for forest species. The flower buds of hibernating

trees differ in their susceptibility to freezing damage. Meth-

ods 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 primordiaretained their viability down to )40C. This strategy (harmo-nized freezing) might help to ensure the survival of trees. In

Arabidopsis, freezing tolerance is associated with the avoid-

ance of damage to the plasmamembrane 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 mem-

brane resealing, and this may be more easily achieved by

incorporating MRI analysis with more conventional biolog-

ical approaches.

THE HOSTPATHOGEN INTERACTION

Plants are challenged by a range of viral, bacterial and fun-

gal 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 data-

sets 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 amajor 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 gener-

ally regarded as being a hardy, drought- and disease-

resistant 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 high-

resolution magic angle spinning NMR spectroscopy for

studying this viral infection. The contrast of T1 and T2weighted images detected differences in the spatial distri-

bution 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 xylo-

philus. Infected trees eventually die as a result of compro-

mised 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 non-

invasive 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 rela-

tionship between the two pathogens, providing new insight

into plantpathogens interactions (Hillnhutter et al., 2011).

SUSTAINING BIODIVERSITY

Biodiversity is threatened by a combination of over-exploi-

tation, 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 pro-

vide a non-invasive assessment of the integrity of a seeds

internal structure, to detect the presence of internal patho-

gens (Kockenberger et al., 2004), to visualize the distribution

of lipids or water within a seed (Ishida et al., 2004; Neu-

berger et al., 2008) and to monitor the physical state of

moisture within a seed as a result of storage at low tem-

peratures (Borompichaichartkul et al., 2005) are all highly

relevant for developing an efficient germplasm conserva-

tion strategy. Systematic comparisons of the structure and

composition of freshly harvested versus stored seeds

(possibly augmented by artificial seed ageing measures)



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 histo-chemical 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 simulta-

neous expression of multiple fluorescent proteins has

resulted in the recognition of some 90 distinguishable col-

ours (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 infor-

mation. 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 immo-

bile) 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 iron-

based 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 Wuyt-

swinkel et al., 1999; Drakakaki et al., 2000; Jiang et al., 2006).

Finally, the switchable chemical exchange saturation trans-

fer (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 rhythmwas maintained even

when the plants were kept under continuous light or dark-

ness. 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 sig-

nal (Liu et al., 2001). Thus the inclusion of MRI data allowed

a linkage between function (water dynamics) and gene

expression. Thewider use ofMRI 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,13C/1H-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 depen-

dent on the degree of Jekyll repression, approving the role

of JEKYLL in nutrient allocation during the process of grain

filling.



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, pre-

senting 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 delineat-

ing 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 under-

stand 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 spectro-

scopy has been widely applied to help understand the

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 devel-

oping seed, and in the quantification of seed storage com-

pounds.

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 themature 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 disad-

vantage 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)

Figure 5. Quantitative imaging of lipid in a living

barley grain. (a) Fragment of a barley spike used

for the magnetic resonance imaging (MRI) anal-

ysis. (b) Longitudinal tissue section showing the

internal structure of the grain. (c) Lipid staining in

a longitudinal tissue section using Sudan/etha-

nol 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 demon-

strating the internal structure of grains/spikewith

resolution of 35 lm. (f) Quantitative map repre-senting 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, peri-

carp. For further details see Neuberger et al.

(2008).



events within a developing seed. In a more recently devel-

oped approach, reliance was placed on the slightly different

resonance frequencies of water and lipids, which could be

exploited using a frequency-selective MRI technique (Neu-

berger et al., 2008). This method shortened the measure-

ment time up to 10-fold, and delivered a spatial resolution

close to the cellular level.

The simultaneous imaging of anatomy and lipid deposi-

tion offers the opportunity to relate lipid accumulation with

seed development. Using this approach in the developing

barley grain revealed concentrated lipid deposition in par-

ticular 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) aswell 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 analyti-

cal opportunities, which are unobtainable by destructive

sampling which induces the wounding response. Chemical

shift imaging has only a minimal requirement for post-pro-

cessing correction, and the acquisition and processing pro-

cedure 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 of1H-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 ima-

ges were acquired at the end of the CSI protocol in order to

topographically relate the spectroscopic data with the cor-

responding tissue structures. As a result, it was apparent

that the spatial distribution of sucrose (as well as of gluta-

mine 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 (Fig-

ure 2c,d). The sucrose concentration gradient was some-

what 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

(a)

(b) (c) (d)

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 depo-

sition 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, endo-

sperm; em, embryo; p, pericarp. For further

details see Hayden et al. (2011).



sensitivity obtainable from small seeds will need some

modification of currently available RF resonators (Neuber-

ger andWebb, 2009). AnotherMRI application formetabolite

imaging is the study byWenzler et al. (2008) of carbohydrate

metabolism involved in forming floral nectar (Anigozanthos

flavidus). These authors combined cyclic J cross-polariza-

tion and 1H spin-echo imaging (a technique implemented

by Heidenreich et al., 1998) to show the localization of13C-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 (Szim-

tenings et al., 2003; Van As, 2007). However, they are difficult

to perform when velocities lie in the lm h)1 range. Thedetection, 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 the13C nucleus (Bax et al., 1983; Rothman et al., 1992). These

pulse sequences provide a range of flexible strategies for the

detection of 13C nuclei and are in generalmore 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 highmagnetic 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 origi-

nated. The 13C/1H-NMR method allowed for the straightfor-

ward co-registration of the structural and the metabolite

images, and therefore the exact identification and localiza-

tion 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 met-

abolically homogeneous. The use of a geHMQC sequence

enabled the detection of 13C alanine, derived by supplying13C 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

(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).



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-tar-

geted experiments, a number of related applications are

conceivable. Experimental plants could be fed with various

13C substrates in order to define the routes by which the

corresponding compounds are taken up, distributed and/or

metabolized. When applying other isotopically labelled nu-

clei (e.g. 15N, 19F, 31P), one needs to consider the shift in

sensitivity of MRI.

Taken together, we argue that dynamicMRI opens up new

perspectives for the non-invasive analysis of metabolic

compartmentalization, metabolic modelling and the identi-

fication of metabolic markers in plants.

ACKNOWLEDGEMENTS

Axel Haase (University of Munich) and Ulrich Wobus (IPK Gater-sleben) are gratefully acknowledged for their support in com-mencing our NMR research on plants. The authors thank GerdMelkus (University of California) and Johannes Fuchs (University ofWurzburg) for dedicated contributions. We also thank Peter M.Jakob (University of Wurzburg), Andrew Webb (University ofLeiden) and Thomas Altmann (IPK Gatersleben) for continuoussupport. We acknowledge funding by the German Federal Ministryof Education and Research, the Deutsche Forschungsgemeinschaftand BayerCropScience.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Magnetic resonance imaging devices used for micro-scopic 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, whichpermitted a three-dimensional visualization of seed anatomy, and inparticular allowed for the measurement of the volume of variousseed organs.Video clip S2. Animated three-dimensional model of lipid distribu-tion in mature barley. High lipid signals (in green) are found in thescutellum and nodule. Lipid in the aleurone is visualized as a bluelayer surrounding the endosperm.Video clip S3. Animated visualization of sucrose allocation withinthe barley caryopsis during grain filling.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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