Tree Physiology 35, 366–375
doi:10.1093/treephys/tpu105
Technical note
A portable NMR sensor to measure dynamic changes
in the amount of water in living stems or fruit and
its potential to measure sap flow
Carel W. Windt1,3 and Peter Blümler2
1Institute
of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Jülich, Jülich, Germany; 2Institute of Physics, University of Mainz, Mainz, Germany;
author ([email protected])
3Corresponding
Received January 31, 2014; accepted November 10, 2014; published online January 15, 2015; handling Editor Kathy Steppe
Nuclear magnetic resonance (NMR) and NMR imaging (magnetic resonance imaging) offer the possibility to quantitatively and
non-invasively measure the presence and movement of water. Unfortunately, traditional NMR hardware is expensive, poorly
suited for plants, and because of its bulk and complexity, not suitable for use in the field. But does it need to be? We here
explore how novel, small-scale portable NMR devices can be used as a flow sensor to directly measure xylem sap flow in a
poplar tree (Populus nigra L.), or in a dendrometer-like fashion to measure dynamic changes in the absolute water content of
fruit or stems. For the latter purpose we monitored the diurnal pattern of growth, expansion and shrinkage in a model fruit
(bean pod, Phaseolus vulgaris L.) and in the stem of an oak tree (Quercus robur L.). We compared changes in absolute stem
water content, as measured by the NMR sensor, against stem diameter variations as measured by a set of conventional point
dendrometers, to test how well the sensitivities of the two methods compare and to investigate how well diurnal changes in
trunk absolute water content correlate with the concomitant diurnal variations in stem diameter. Our results confirm the existence of a strong correlation between the two parameters, but also suggest that dynamic changes in oak stem water content
could be larger than is apparent on the basis of the stem diameter variation alone.
Keywords: bean, compact, growth, MRI, oak, poplar, portable mobile, transport, water content.
Introduction
Ever since nuclear magnetic resonance (NMR) was discovered,
more than half a century ago, researchers have recognized its
potential to study plants and fruit (Shaw and Elsken 1956, Van
Putte and Van den Enden 1973, Gusta et al. 1975). The most
important feature of NMR for plant sciences is that it makes it
possible to non-invasively measure the presence and mobility
of protons (i.e., the nucleus of the hydrogen isotope 1H) in
water, oil and fat, without any need for tracers. This property
has been successfully exploited in a wide variety of applications. Typical examples are the imaging of anatomy (Hinshaw
et al. 1979), the effects of illnesses in growing plants and fruit
(Clark et al. 1997, Umebayashi et al. 2011), water content and
cavitation events in stems (Scheenen et al. 2007, Choat et al.
2010), growth of roots in soils (Jahnke et al. 2009, Hillnhütter
et al. 2012), and xylem and phloem sap flow imaging in stems,
roots and fruits (Köckenberger et al. 1997, Scheenen et al.
2002, Windt et al. 2006, 2009). For a more comprehensive
overview, please see the review by Borisjuk et al. (2012).
Nuclear magnetic resonance has become a well-proven
method, but is still far from a standard research tool in botany. The most important factors preventing this are the cost,
size and complexity of the NMR equipment. The most troublesome component is the NMR magnet, which by necessity is
at the heart of every NMR scanner. These days standard NMR
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An NMR sensor to measure changes in the amount of water in living stems or fruit 367
experiments are conducted on superconducting, tube-shaped
magnets. To insert or even handle an intact plant inside the
bore of such magnets, the diameter of the bore must be wider
than the shoot or crown, and if the plant is not long enough to
reach the magnet's center, wider than the pot as well. Large
superconducting magnets are not only expensive to buy, but
also costly to run and maintain. Most models require continuous topping-up with liquid helium, continuous cooling, or both.
In addition to that, large scanners also need powerful, high-end
electronics to drive them.
Nuclear magnetic resonance can become much more affordable when the samples are small, and when neither imaging
nor high field strengths are needed. When low to moderate
field strengths (0.1–1 T) are sufficient, the magnet can be constructed from permanent magnets that do not require electrical
power or cryogens to function. Spatial- and flow-encoding typically require strong gradient amplifiers and coils. When imaging
is not needed two of the high-powered gradient amplifiers can
be omitted, as well as the accompanying gradient coils; when
flow measurements are also not needed the third and last
remaining gradient amplifier and coil can be omitted as well.
Nuclear magnetic resonance relaxometry is an example of a
method that in many cases meets these reduced requirements.
It is not directly a spatially resolving method; however, average sizes of restrictions (e.g., microscopic pore size distributions) can be estimated from models fitted to the relaxometric
data. Normally it is used for determining the water and dry
matter content or oil and solid fat content of samples varying
from test tubes to entire animals (van Duynhoven et al. 2010).
Relaxometry revolves around two elemental properties of the
NMR, namely that the signal intensity scales linearly and quantitatively with the number of protons in the coil, and that the
relaxation behavior of the NMR signal depends on the mobility
of the protons, as well as the physicochemical properties of
their surroundings (van Duynhoven et al. 2010). Most commercial moderate-field NMR relaxometers are small enough to fit on
a table top, but are by no means portable. The magnets typically are too heavy to handle by hand, and due to their box-like
construction they usually do not offer optimal access for intact
plants. The associated electronic components, due to their size,
weight and power requirements, also do not invite mobile use.
That does not mean that it is impossible though, as was exemplified by Geya et al. (2013) who went as far as to construct an
electric lorry to be able to haul a full-sized magnetic resonance
imaging (MRI) console with a custom-built permanent magnet
into the field for a relaxometric study on developing pear fruit.
In order to transform an NMR scanner into a device that
is suitable for use in the field, two main factors need to be
addressed that determine the size and portability of an NMR
system: the electronic components (spectrometer, r.f. amplifier
and eventual gradient amplifiers) and the magnet. In both areas,
great progress has been made in the last decade. Cell-phone
technology has made powerful integrated circuitry available to
hardware builders, giving rise to a generation of ever smaller
and more energy-efficient spectrometers that can be run on
battery power. These are now commercially available, and
designs to build do-it-yourself versions of such spectrometers
have even been released to the public domain (Takeda 2011).
Nuclear magnetic resonance magnets have been revolutionized as well. New ground-breaking magnet designs have been
introduced that offer free access to objects, either by using a
single-sided design also known as the NMR-MOUSE (Blümich
et al. 2011), by using a modified Halbach tube structure that
is closed, but can be opened up to accept the plant (NMRCUFF) (Windt et al. 2011), or by using a more conventional, but
scaled-down and light-weight C-shaped magnet design that is
permanently open from one side (Rascher et al. 2011). When
the magnet's isocenter is freely accessible there is no more
need for the bore or the air gap of the magnet to be wider than
the plant's crown. Instead, it only needs to be wider than the
plant part of interest. This makes it possible to scale down the
size of the magnet tremendously, to the point that it becomes
portable, can simply be slid over objects such as branches or
stems and will even fit in between the leaves.
In this study we explore how simple, low-field relaxometryinspired NMR devices can be used in a sensor-like fashion to
monitor sap flow and dynamic changes in the amount of water
in the intact tree or fruit. We do so, firstly by providing proof of
principle that small-scale, non-spatially resolving NMR devices
have the potential to be used to measure xylem sap flow in the
intact tree. Secondly, we illustrate how even simpler, low-end
NMR sensors can be used to monitor net water uptake and
diurnal changes in the amount of water in developing fruit, with
a high time resolution and if desired continuously for weeks
without interruption. We further demonstrate how such measurements can be employed to assess a fruit's apoplastic connectivity. Thirdly, we present an experiment in which we monitor
the diurnal pattern of expansion and shrinkage of an oak tree
by means of an NMR sensor and a set of point dendrometers,
to test how well the sensitivities of the two methods compare
and to investigate how well diurnal changes in trunk absolute
water content correlate with the concomitant diurnal variations
in stem diameter.
Materials and methods
Plant material and handling: poplar
A potted 1.75-m tall poplar tree (Populus nigra L., diameter
∼7 mm) was grown in a greenhouse under ambient light, supplemented with additional light (Philips SON-T Agro 400 W,
Philips, Osnabrück, Germany) whenever the light i­ntensity
fell <390 µmol m−2 s−1, resulting in a lower light limit of
∼200 µmol m−2 s−1. Day/night conditions: 16 h light/8 h dark,
22 °C/24 °C, relative humidity (RH) 50/70%. The tree was
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368 Windt and Blümler
kept well watered by a drip irrigation system. The day prior to
the flow measurements the NMR-CUFF was mounted at the
base of the trunk, after which the tree was moved into the lab
(22 °C, 250 µmol m−2 s−1 PAR, Philips SON-T Agro 400 W).
Plant material and handling: bean
Plants of French bean (Phaseolus vulgaris L. ‘Fadenlose Shiny’)
were grown from seeds in standard potting soil, first in 1.7 l
pots, then after 2 months repotted into 7 l pots using the same
substrate. The plants were grown in a greenhouse under ambient light, supplemented with additional light (Philips SON-T Agro
400 W) whenever the light intensity fell <390 µmol m−2 s−1,
resulting in a lower light limit of ∼200 µmol m−2 s−1. Day/night
conditions: 16 h light/8 h dark, 24 °C/22 °C, RH 50/70%.
For the experiment a plant with several fertilized inflorescences was selected and placed into a climate chamber
(Sanyo MLR-350H). Day/night conditions: 16 h day/8 h night,
23 °C/23 °C, RH 70%, 250 µmol m−2 s−1 PAR. The NMR magnet
was placed on an adjustable stand next to the plant, inside the
climate chamber. The plant was left to acclimate in the climate
chamber for several days before the measurement. At the start
of the measurements, a vigorously growing, 1-week-old bean
pod was selected and gently placed in the center of the NMR
probe, where the pod was monitored for a period of 2 weeks.
Plant material and handling: oak
Measurements were conducted on the main stem of a 3-yearold oak tree (Quercus robur L.). The tree was acquired from a
nursery 2 years prior to the experiment and subsequently grown
in the open air in a 10-l container. The containers were filled
with potting soil and fertilized with a slow releasing NPK plus
magnesium mix (Basacote Plus 6M, COMPO, Deinze, Belgium).
The trees were kept well watered by means of an automatic
drip-watering system. In late spring, 1 month before the start
of the experiment, the trees were taken to the laboratory and
allowed to adjust to the experimental conditions. The trees
were subjected to a 16-h day/8-h night cycle, temperatures of
20–23 °C during the day and 19–20 °C at night. The RH varied between 40 and 70% (not controlled). Two metal halide
lamps (Philips IP65+SON-T Agro 400 W, Philips, Osnabrück,
Germany) p
­ rovided 300 µmol m−2 s−1 PAR at canopy level.
A C-shaped NMR magnet was mounted around the tree
at a height of 80 cm. The magnet was supported by a tripod
(Manfrotto 117b, Cassola, Italy), equipped with a heavy duty ball
head (Manfrotto #168, Cassola, Italy), allowing the magnet to be
tilted and rotated in all directions. At mounting height the tree had
a diameter of 12 mm.
Variations in stem diameter were measured continuously
with linear variable displacement transducers (LVDTs; Model
DF5.0, Solartron Metrology, Leicester, UK). The dendrometers
were supported by a custom-made stainless-steel frame that
does not require a temperature correction, as developed and
Tree Physiology Volume 35, 2015
described by Steppe and Lemeur (2004). The LVDTs were
attached to the tree at a distance of 20 cm above and below
the magnets’ isocenter, as close to the magnet as possible
without influencing the LVDTs or affecting the homogeneity
of the magnetic field. All sensor signals were logged at 10 s
intervals and 5 min means were stored using a data logger
(CR1000, Campbell Scientific, Bremen, Germany).
NMR hardware and methods: velocimetry
For the xylem flow experiment in poplar a small, home-built 0.57T
NMR-CUFF magnet with a 3-cm bore was used (Figure 1a).
A unique feature of the NMR-CUFF is that, despite the closed
ring structure of the Halbach magnet which is at the base of its
design, it is hinged and can be split open without force (Windt
et al. 2011). This way it could be easily mounted on the base of
the poplar tree without damage. Before placing the NMR-CUFF,
an 11-turn, 10-mm diameter solenoidal r.f. coil was wound around
the stem with the help of a Teflon former. A hinged, custom-built
630 mT/m gradient set was then fitted around the r.f. coil. The
NMR-CUFF was subsequently clamped around the r.f. coil and
the gradient assembly (Figure 1b) and placed on a wooden support. With an outer diameter of 9.5 cm and a height of 12 cm, the
sensor could remain remarkably compact.
During NMR velocimetry a sample is excited by means of
a sequence of r.f. pulses, generating a signal that is directly
proportional to the amount of liquid water in the probe. This
signal decays in milliseconds to seconds, depending on the
tissue. During this time displacement encoding is done. Water
is electromagnetically labeled for position along the Z axis (no
need for chemical labeling agents), then allowed to move about
for a certain period (here: 20 ms), after which the changes
in position along the Z axis are read out. The velocity spectrum (or propagator) of water moving along Z is thus recorded
(Scheenen et al. 2000a, 2000b). Displacements in other
directions than along Z, such as radial flow and exchange, do
not become visible in the velocity spectrum.
For the flow measurement a Bruker Avance spectrometer console was used, equipped with a 500 W BLAX r.f. amplifier and
a single BAFPA 40 gradient amplifier (BRUKER, Rheinstetten,
Germany). Experimental parameters: repetition time 2.5 s, echo
time 3.5 ms, 32 echoes, 8 averages, slice thickness 1.3 mm,
spectral width 200 kHz. The scan time was ∼11 min. The propagator was acquired by sampling q-space equidistantly, with gradient pulses from plus to minus 0.63 T/m in strength and 4 ms
in duration, in combination with a flow labeling time of 20 ms.
NMR hardware and methods: measuring absolute
water ­content
For the bean and oak tree experiments a home-built C-shaped
magnet was employed. The field strength of the magnet was
0.26 T over a 3 cm air gap; the magnet weighed 4.5 kg. In both
cases a 13-turn solenoidal r.f. coil with an inner diameter of
An NMR sensor to measure changes in the amount of water in living stems or fruit 369
Figure 1. Mobile NMR magnets. For the flow measurement in poplar the prototype of a 0.57-T NMR-CUFF-type magnet was used (a), fitted with
a 10-mm diameter solenoidal r.f. coil on a Teflon spindle and a hinged, custom-built 630 mT/m gradient set (b). The NMR-CUFF could simply be
opened and clamped around the coil and gradient assembly. To handle large trees an NMR-CUFF prototype with a 15.6-cm bore was constructed,
here shown around a tree trunk of 10 cm in diameter (c). For reasons of mobility the magnet is placed on wheels (not shown). For the bean pod
and oak experiments a 0.26-T C-shaped magnet with a 15-mm r.f. coil was employed (d). The magnet itself is not visible because it is enclosed in
a housing to provide thermal isolation. In between the poles of the C-shaped magnet the removable copper r.f. shielding of the probe can be seen;
for samples such as the oak tree this can be opened up to give free access to the magnet's isocenter.
15 mm was used for excitation and detection. The coils were
hand-wound around the trunks with the aid of a split Teflon coil
former. In order to reduce magnetic field drift due to changes
in magnet temperature, the magnet was equipped with an
accurate regulated heating module and placed in thermally
insulated aluminum casing (Figure 1d). The temperature was
set to 25 °C, a few degrees above the highest expected day
temperature.
The NMR probe was driven with a standard Kea II spectrometer (Magritek, Wellington, New Zealand), equipped with
a built-in 100 W r.f. amplifier. For each data point a CPMG type
measurement was run, with a repetition time of 7.5 s, 3000
echoes, an inter-echo time of 750 µs, eight complex points
per echo, 32 averages and a spectral width of 100 kHz; the
total scan time for each point was 4 min. In order to mimic
an experiment that could be done using even the most basic
of spectrometers and that would allow unsupervised, real-time
data processing, we chose to not use all acquired echoes but
only employ the first ones, averaging all echoes between 0 and
25 ms. This approach maximizes signal to noise, avoids fitting
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370 Windt and Blümler
errors that might easily occur in environments with changing
environmental conditions (Gultekin and Gore 2005) and was
shown to yield an excellent correlation with probe (r.f. coil)
water content. The temperature of the tree stem in the NMR
sensor was continuously monitored, as was the temperature
of the spectrometer. The slight differences in amplitude of the
NMR signal that might result from temperature differences of
the object (due to a shift in the Boltzmann equilibrium) and
temperature-induced differences in the signal amplification factor of the spectrometer were subsequently corrected for by
applying an appropriate temperature correction factor. The corrected amplitude that is measured in this fashion scales linearly
with the amount of liquid water inside the sensitive volume of
the r.f. coil in the NMR probe head.
To avoid confusion with the term water content, which by
definition is a relative unit relating either to the total mass or
to the total volume of the sample (both of which are values
that the NMR sensor cannot measure directly and that cannot
be assumed to remain constant in a growing organ), we here
choose to express changes in amplitude as changes in absolute water content or absolute water content increment. The
absolute water content increment (ΔWCabs) is calculated and
expressed as follows:
 A(t = t )

∆WCabs = 
− 1 × 100%
 A(t = 0)

where A(t = 0) is the amplitude at time 0 and A(t = t) the amplitude
at time t. Please note that here t denotes the time between the
two measurements, not the time between excitation and signal
acquisition as it is usually defined in the NMR community.
Results and discussion
Magnet versus object size
In most cases, permanent magnet designs can be scaled up to
handle objects of an arbitrarily large diameter. The small diameter of the poplar tree made it possible to use an extremely
compact and light-weight magnet, with a bore of 3 cm in diameter and a weight of merely 3.1 kg.
To be able to measure full-grown trees, a prototype of a
much larger NMR-CUFF with a bore diameter of 15.6 cm and
a maximum useable diameter of 10 cm was constructed as
well (Figure 1c). Unfortunately, if the size of the bore (or pole
gap) of a magnet is doubled, the weight of the magnet will not
simply double as well, but scale with the power of three. In
practice this means that, even when machines such as tractors would be available to transport the magnet, it would be
challenging to handle trees that are much larger than this. In
the example shown here, we tackled the problem by mounting
the magnet on a heavy duty aluminum table, fitted with large
wheels to allow the device to be moved around over uneven
Tree Physiology Volume 35, 2015
surfaces. A further drawback is that larger magnet bores and
larger objects call for larger (flow) imaging gradients, larger r.f.
coils, and therefore, high-powered r.f. and gradient amplifiers.
Such amplifiers typically weigh between 20 and 50 kg each
and can no longer be run from battery power.
Small object sizes, on the other hand, do not pose a problem. As long as the r.f. coil that is used for the excitation of the
sample and the reception of the NMR signal closely fits the
object of study, be it a tree trunk or a pine needle, the signalto-noise ratio of a measurement can be expected to remain
the same.
Measuring xylem sap flow
So far NMR velocimetry, also known as NMR flow imaging or
NMR flowmetry, has very much relied on imaging and high
magnetic field strengths. The high field strength serves to
maximize the signal-to-noise ratio of a measurement, and the
spatial resolution to allow for separating flow containing voxels
from the ones that do not contain flow. Both properties are
helpful when attempting to detect small amounts of flowing
water in the presence of large amounts of stationary water. The
amount of flowing sap, in relation to the amount of stagnant
water in the rest of the plant part under investigation, typically
is very small (Scheenen et al. 2000b, Windt et al. 2006). We
here take a different approach and explore the possibility to
measure xylem sap flow in the stem of a young poplar tree
by means of a low field, non-spatially resolving NMR sensor
(Figure 1a and b), the NMR-CUFF (Windt et al. 2011).
By means of this setup a velocity spectrum could readily
be obtained (Figure 2). A clear asymmetric deviation of the
velocity spectrum with respect to zero velocity can be seen on
the right-hand side, i.e., in an upward direction, but not on the
left-hand side. If we may assume that the left-hand side only
represents stationary diffusing water, which is to say, that the
volume of downward moving phloem sap relative to the volume
of upward moving xylem sap can be assumed to be negligible
(Windt et al. 2006) and that in the xylem no downward flow
was present, then the ‘non-flow’ side of the velocity spectrum
can be mirrored with respect to the Y axis and subtracted from
the ‘flow’ side of the velocity spectrum, to yield the velocity
spectrum of the flowing water as well as that of the stagnant
water (Figure 2b) (Scheenen et al. 2000a). The area under the
respective graphs then directly and quantitatively relates to the
amount of flowing and stagnant water in the tree (Scheenen
et al. 2000b).
Due to the large amount of flowing water in the tree, in combination with the high sap flow velocities, it was remarkably
straightforward to detect the flowing xylem sap and to separate
it from the stationary water. A single measurement thus could
be done in 11 min, which is about twice as fast as a comparable
spatially resolved measurement on a high-end stationary imager
(Scheenen et al. 2000b, Windt et al. 2006). The relative ease
An NMR sensor to measure changes in the amount of water in living stems or fruit 371
Figure 2. Nuclear magnetic resonance velocimetry: xylem sap flow measurement in the stem of an intact poplar tree (P. nigra). (a) Cumulative
velocity spectrum (propagator) of all water in a cross-section of the stem. The asymmetry in the velocity spectrum was generated by directional
motion (flow), whereas stagnant but randomly diffusing water became visible as an assumingly symmetric Gaussian shape centered around zero
(b, gray line with open dots). Xylem sap flow, moving upward from roots to leaves, as expected only exhibits positive velocities (b, black line with
closed dots).
with which in this example xylem sap flow could be detected
shows good promise for the use of low-end NMR devices to
measure sap flow and derivative parameters such as plant water
use in the field. While the results presented here constitute proof
of principle that xylem sap flow can be detected irrespective
of spatial resolution, it should however be emphasized that the
accuracy of the method for now remains untested.
Monitoring fruit water content: bean pod
One of the most basic applications of an NMR sensor is to use
the device as a water detector. If NMR parameters are suitably
chosen, the amplitude of the NMR signal scales linearly and
quantitatively with the total amount of liquid water inside the
sensitive region of the sensor (Levitt 2008). Such measurements are robust and fast, and can be surprisingly useful: they
provide a unique means to directly but non-invasively monitor the amount of water in a plant, measure expansion growth
(here defined as growth by a net uptake of water), or by taking dynamic changes in the amount of water as a proxy for
changes in water potential, plant water status. To illustrate this
point we present two examples.
The first example is an experiment in which the dynamic
accumulation and loss of liquid water are monitored in a developing bean pod (P. vulgaris) and related to the fruit's growth
and apoplastic connectivity. The pod was inserted into an
NMR sensor, in this case consisting of a home-built, C-shaped
magnet with a 3 cm air gap (Figure 1d). This type of magnet
provides a lower field strength per unit of magnet mass than
the NMR-CUFF, but has the advantage that it is permanently
open from one side, thus providing easier access to plants
(not apparent in the figure due to the shielding of the probe).
After insertion an automated measurement sequence was run,
autonomously acquiring a reading every 4 min over a period
of 2 weeks. The resulting growth curve is shown in Figure 3.
It should be noted that during the course of the experiment
the bean pod not only grew in thickness, but also in length:
from 9.5 cm at the start of the experiment to 20 cm after
2 weeks. The 370% increase in absolute water content that
is visible in the graph thus does not represent the expansion
growth of the pod as a whole, but that of a 15-mm thick crosssectional slice of it. The total increase in the amount of water
in the pod thus will have been larger still. Towards the end of
the second week, the amount of water in the pod was slowly
decreasing.
The rapid expansion caused the pod to slightly shift position during the measurements. The result of such a shift can,
for instance, be seen around Day 12. At that moment, the pod
already had developed sizeable beans. When at such a stage
the narrow ‘field of view’ of the NMR scanner would shift along
the length of the pod, the apparent changes in the amount of
water in the pod could be significant. In future applications this
could be avoided, for example, by using an r.f. coil that is long
enough to contain the entire pod. In the current setup, however, shifts could not be prevented. For this reason we here
focus on the timeframes in which the position of the pod could
be assumed to be stable (Figure 3, detail graphs day/night
transitions 3, 7, 10 and 14).
The four detailed 24 h graphs exhibited a number of interesting features. Throughout the whole growth period the pod's
absolute water content was strongly affected by the light and
dark cycles. At night the net water uptake of the pod was always
larger than that during the day. Towards the end of Week 2 and
beyond, the pod not only lost water at the moment that the
lights were switched on, but also began to exhibit a net loss of
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372 Windt and Blümler
Figure 3. The dynamics of water accumulation and water loss in a developing bean pod. Shown is the development of a young bean pod, measured
over a period of 2 weeks by means of a custom-built portable NMR scanner. The bean pod was 7 days old at the start of the measurement. Four
day/night/day transitions are shown in detail, at night number 3, 7, 10 and 14 (indicated with dashed lines in the main graph). The night periods
are indicated with dark gray bars. The amount of liquid water in the 15-mm long section of bean pod that is enclosed by the NMR coil in the probe
head is expressed as the absolute water content increment, in percent relative to the value at t = 0.
water throughout the day, and no longer exhibited a net water
uptake at night. Sudden, but mild changes of the hydrostatic
pressure in the apoplast of the plant, such as that caused by the
lights of the climate chamber switching on and off, were transmitted to and equilibrated by the pod in <2 h. The pod is known
to lose water by transpiration (Pate et al. 1985), but it is hard
to imagine that this could be sufficient to cause such significant
changes to the amount of water in the pod, especially when it
is considered that the pod was partially shielded from the light
by the magnet and the sample holder, and also experienced a
significant net uptake of water whenever the lights were turned
off. We therefore conclude that, throughout the 2-week growth
period, the bean pod was apoplastically well connected, causing it to instantly experience the same diel changes in water
potential as vegetative parts of the plant.
This example illustrates how a simple portable NMR device
can be used to measure the effects of sap flow toward fruit,
non-invasively and with good accuracy and temporal detail. As
simple as the measurement is, it offers a number of interesting
possibilities. It could be combined with conventional means to
assess fruit growth and fruit evaporation (such as gas exchange
measurements and phloem girdling techniques) to not only
determine the fruits apoplastic connectivity, but to also get a
handle on its entire phloem and xylem sap flow balance.
Tree Physiology Volume 35, 2015
Monitoring stem absolute water content in oak
The second example featuring the NMR sensor in the role of a
water detector is an experiment in which the diurnal pattern of
growth, expansion and contraction was monitored in the stem
of an oak tree. Usually such studies are done by means of pointor band-type dendrometers. Sensors of this type have become
the tool of choice to study dynamic tree growth responses to
various environmental factors (Molz and Klepper 1973, Zweifel
and Häsler 2001, Drew and Downes 2009). They are inexpensive and can be left on the tree for months. However, trunk
diameter itself often is not the parameter of interest. Rather, it
is used as a proxy to estimate parameters such as stem water
content or stem water potential (Zweifel et al. 2000, 2001),
sap flow dynamics (Steppe and Lemeur 2004, Steppe et al.
2006) or as an indicator for the onset of abiotic stresses. In
this demonstrator experiment, we monitor the diurnal pattern
of water uptake and loss, stem diameter growth and shrinkage,
in an oak tree stem by means of an NMR sensor and a set of
point dendrometers, to compare the sensitivity of the two types
of measurements and to address the question of whether stem
diameter differences as measured by dendrometers accurately
reflect changes in trunk water content.
The two dendrometers revealed that over the 6-day period
the tree was steadily growing, but despite the fact that the tree
An NMR sensor to measure changes in the amount of water in living stems or fruit 373
was kept well watered and was growing under moderate conditions, only exhibited growth at night (Figure 4a). During the
day the stem diameter remained largely unchanged. In order to
be able to more directly compare the diameter variation curves
against the NMR sensor readings, we calculated the average
relative cross-sectional area on the basis of the diameter variation data (Figure 4b).
The absolute water content as measured by NMR largely
followed the same pattern as the diameter variation or the
average cross-sectional area increment. Growth became visible as a marked increase in the absolute water content during
the night, whereas during the day periods it remained constant. During the 6 days of observation the increase in the
stem cross-sectional area was 3.9%. This was almost double
the value that was found for the increase in the amount of
water in the stem, 2.1%, nicely illustrating the fact that in oak,
secondary growth is as much a function of dry matter deposition and wood formation as it is of the division and expansion of water containing living cells. After harvest the stem
Figure 4. Stem diameter variation and stem absolute water content
in oak, measured over a 6-day period. In a 3-year-old oak tree stem
diameter variations were measured by means of a set of LVDT point
dendrometers, mounted above and below the NMR sensor (a). From
the diameter variations the cross-sectional area increment at the position of the NMR sensor was estimated, and plotted together with the
absolute water content increment as measured by means of NMR (b).
The change in both parameters is given in percent relative to the value
at time 0. Night periods are indicated with dark gray bars.
piece under observation was found to have a moisture content
of 46.6%.
Nuclear magnetic resonance is not considered to be an
exceptionally sensitive method, especially at low magnetic field
strengths. Yet, the NMR sensor seemed to be even more sensitive in picking up small variations in stem absolute water content than were the LVDTs. At the moments that the lights were
switched off marked jumps in absolute water content were
observed, whereas at the moments that the lights were turned
on sharp decreases were measured. In the first case absolute
water content, on average, increased by 0.5% in a timespan of
55 min, whereas when the light was turned on it, on average,
decreased by 0.6% in 46 min. A similar pattern of expansion
and contraction was recorded by the LVDT mounted above the
NMR sensor, closest to the crown, but with a reduced amplitude, and was not observed at all by the LVDT mounted below.
A large number of experimental and modeling studies confirm the existence of a direct, causal relationship between
changes in stem diameter and stem water content (Simonneau
et al. 1993, Zweifel et al. 2000, 2001, Sternberg and Shoshany
2001, Zweifel and Häsler 2001, Steppe et al. 2012), but due
to experimental difficulties only two studies, both involving noninvasive NMR, have been able to directly and non-invasively
relate changes in stem water content to diameter variations in
the same intact, living stem. The first is a study by Reinders
et al. (1988), who used NMR to measure water content in a
herbaceous vine (cucumber) and showed that changes in the
amount of water in the stem closely match the changes in its
diameter. More recently De Schepper et al. (2012) used MRI
in combination with dendrometer readings to not only confirm
the existence of a close link between stem water content and
stem diameter, but also to quantify the contribution of water
from different tissues in the stem to the observed diurnal stem
diameter variations. They concluded that stem diameter variations are mainly caused by changes in the amount of water in
elastic bark tissues, excluding the young conducting phloem
and the cambium.
In the current exploratory study, we demonstrate that the
NMR sensor has the potential to be used as an NMR-based
dendrometer in its own right. By using a compact, low-field
portable NMR sensor instead of a fully fledged high-end MRI
scanner we lose the ability to visualize in exactly what tissue
water is lost or taken up, but make up for that by the fact that
the device is portable, suitable for use on site in the field or
forest, and can obtain data with a high temporal resolution and
with an accuracy that in the current example surpassed that
of conventional dendrometers. The interpretation of the resulting data is relatively simple because the device uniquely measures the amount of liquid water in the stem. The comparison
between the diurnal stem diameter variations and the diurnal
stem absolute water content variations again confirms the existence of a strong correlation between the two parameters, but
Tree Physiology Online at http://www.treephys.oxfordjournals.org
374 Windt and Blümler
also suggests that the dynamic changes in the amount of water
in the stem might be much larger than is apparent on the basis
of the stem diameter variations alone.
We here used the NMR sensor to measure liquid water. By
applying NMR relaxometric principles it will however also be
possible to measure proton-bearing solids and to discriminate
between liquids and solids on the basis of proton mobility
(Musse et al. 2013). In the near future, we expect to exploit
this property in the NMR sensor to non-invasively monitor
changes in fresh and dry weight in the living plant or fruit.
Acknowledgments
The authors thank A. Dahmen and J. Kochs for their help in
the design and construction of the magnet prototypes and the
associated hardware. They thank Dr H. Van As (Wageningen
University, The Netherlands) for the use of the Bruker spectrometer in his lab, J. Philippi (Wageningen University) for his
contribution in the construction of the r.f. coil that was used
for the bean pod experiment and Prof. Kathy Steppe (Ghent
University, Belgium) for lending out the LVDT sensors and a
data logger. Prof. U. Schurr and Dr S. Jahnke are gratefully
acknowledged for making the presented research possible.
Conflict of interest
None declared.
Funding
This research was in part supported by the Helmholtz Initiative
and Networking fund VIP-NMR, project number VH-VI-226.
References
Blümich B, Casanova F, Dabrowski M et al. (2011) Small-scale instrumentation for nuclear magnetic resonance of porous media. New J
Phys 13:015003.
Borisjuk L, Rolletschek H, Neuberger T (2012) Surveying the plant's
world by magnetic resonance imaging. Plant J 70:129–146.
Choat B, Drayton WM, Brodersen C, Matthews MA, Shackel KA,
Wada H, McElrone AJ (2010) Measurement of vulnerability to
water stress-induced cavitation in grapevine: a comparison of four
techniques applied to a long-vesseled species. Plant Cell Environ
33:1502–1512.
Clark CJ, Hockings PD, Joyce DC, Mazucco RA (1997) Application of
magnetic resonance imaging to pre- and post-harvest studies of
fruits and vegetables. Postharvest Biol Technol 11:1–21.
De Schepper V, van Dusschoten D, Copini P, Jahnke S, Steppe K
(2012) MRI links stem water content to stem diameter variations in
transpiring trees. J Exp Bot 63:2645–2653.
Drew DM, Downes GM (2009) The use of precision dendrometers in
research on daily stem size and wood property variation: a review.
Dendrochronologia 27:159–172.
Geya Y, Kimura T, Fujisaki H, Terada Y, Kose K, Haishi T, Gemma H,
Sekozawa Y (2013) Longitudinal NMR parameter measurements of
Tree Physiology Volume 35, 2015
Japanese pear fruit during the growing process using a mobile magnetic resonance imaging system. J Magn Reson 226:45–51.
Gultekin DH, Gore JC (2005) Temperature dependence of nuclear
magnetization and relaxation. J Magn Reson 172:133–141.
Gusta LV, Burke MJ, Kapoor AC (1975) Determination of unfrozen
water in winter cereals at subfreezing temperatures. Plant Physiol
56:707–709.
Hillnhütter C, Sikora RA, Oerke E-C, van Dusschoten D (2012) Nuclear
magnetic resonance: a tool for imaging belowground damage
caused by Heterodera schachtii and Rhizoctonia solani on sugar
beet. J Exp Bot 63:319–327.
Hinshaw WS, Bottomley PA, Holland GN (1979) A demonstration of
the resolution of NMR imaging in biological systems. Experientia
35:1268–1269.
Jahnke S, Menzel MI, Van Dusschoten D et al. (2009) Combined MRI–
PET dissects dynamic changes in plant structures and functions.
Plant J 59:634–644.
Köckenberger W, Pope JM, Xia Y, Jeffrey KR, Komor E, Callaghan PT
(1997) A non-invasive measurement of phloem and xylem water
flow in castor bean seedlings by nuclear magnetic resonance microimaging. Planta 201:53–63.
Levitt MH (2008) Spin dynamics: basics of nuclear magnetic resonance. John Wiley and Sons, Ltd, Chicester.
Molz FJ, Klepper B (1973) On the mechanism of water-stress-induced
stem deformation. Agron J 65:304–306.
Musse M, De Franceschi L, Cambert M, Sorin C, Le Caherec F, Burel
A, Bouchereau A, Mariette F, Leport L (2013) Structural changes in
senescing oilseed rape leaves at tissue and subcellular levels monitored by nuclear magnetic resonance relaxometry through water
status. Plant Physiol 163:392–406.
Pate JS, Peoples MB, van Bel AJE, Kuo J, Atkins CA (1985) Diurnal
water balance of the cowpea fruit. Plant Physiol 77:148–156.
Rascher U, Blossfeld S, Fiorani F et al. (2011) Non-invasive approaches
for phenotyping of enhanced performance traits in bean. Funct Plant
Biol 38:968–983.
Reinders JEA, Van As H, Schaafsma TJ, Sheriff DW (1988) Water balance in cucumis plants measured by NMR II. J Exp Bot 39:1211–1220.
Scheenen TWJ, van Dusschoten D, de Jager PA, Van As H (2000a)
Microscopic displacement imaging with pulsed field gradient turbo
spin-echo NMR. J Magn Reson 142:207–215.
Scheenen TWJ, van Dusschoten D, de Jager PA, Van As H (2000b)
Quantification of water transport in plants with NMR imaging. J Exp
Bot 51:1751–1759.
Scheenen TWJ, Heemskerk A, de Jager PA, Vergeldt FJ, Van As H
(2002) Functional imaging of plants: a nuclear magnetic resonance
study of a cucumber plant. Biophys J 82:481–492.
Scheenen TWJ, Vergeldt FJ, Heemskerk AM, Van As H (2007) Intact plant
magnetic resonance imaging to study dynamics in long-distance sap
flow and flow-conducting surface area. Plant Physiol 144:1157–1165.
Shaw TM, Elsken RH (1956) Moisture determination, determination of
water by nuclear magnetic absorption in potato and apple tissue.
J Agric Food Chem 4:162–164.
Simonneau T, Habib R, Goutouly J-P, Huguet J-G (1993) Diurnal
changes in stem diameter depend upon variations in water content:
direct evidence in peach trees. J Exp Bot 44:615–621.
Steppe K, Lemeur R (2004) An experimental system for analysis of the
dynamic sap-flow characteristics in young trees: results of a beech
tree. Funct Plant Biol 31:83–92.
Steppe K, De Pauw DJW, Lemeur R, Vanrolleghem PA (2006) A mathematical model linking tree sap flow dynamics to daily stem diameter fluctuations and radial stem growth. Tree Physiol 26:257–273.
Steppe K, Cochard H, Lacointe A, Améglio T (2012) Could rapid diameter changes be facilitated by a variable hydraulic conductance?
Plant Cell Environ 35:150–157.
An NMR sensor to measure changes in the amount of water in living stems or fruit 375
Sternberg M, Shoshany M (2001) Aboveground biomass allocation
and water content relationships in Mediterranean trees and shrubs
in two climatological regions in Israel. Plant Ecol 157:173–181.
Takeda K (2011) Chapter 7—Highly customized NMR systems
using an open-resource, home-built spectrometer. In: Graham
AW (ed) Annual reports on NMR spectroscopy. Academic Press,
London, UK, pp 355–393.
Umebayashi T, Fukuda K, Haishi T, Sotooka R, Zuhair S, Otsuki K
(2011) The developmental process of xylem embolisms in pine wilt
disease monitored by multipoint imaging using compact magnetic
resonance imaging. Plant Physiol 156:943–951.
van Duynhoven J, Voda A, Witek M, Van As H, Graham AW (2010)
Time-domain NMR applied to food products. In: Graham AW (ed)
Annual reports on NMR spectroscopy. Academic Press, London, UK,
pp 145–197.
Van Putte KPAM, Van den Enden J (1973) Pulse NMR as a quick
method for the determination of the solid fat content in partially
crystallized fats. J Phys E Sci Instrum 6:910–910.
Windt CW, Vergeldt FJ, De Jager PA, Van As H (2006) MRI of longdistance water transport: a comparison of the phloem and xylem
flow characteristics and dynamics in poplar, castor bean, tomato
and tobacco. Plant Cell Environ 29:1715–1729.
Windt CW, Gerkema E, Van As H (2009) Most water in the tomato
truss is imported through the xylem, not the phloem: a nuclear magnetic resonance flow imaging study. Plant Physiol 151:830–842.
Windt CW, Soltner H, Dusschoten Dv, Blümler P (2011) A portable
Halbach magnet that can be opened and closed without force: the
NMR-CUFF. J Magn Reson 208:27–33.
Zweifel R, Häsler R (2001) Dynamics of water storage in mature subalpine Picea abies: temporal and spatial patterns of change in stem
radius. Tree Physiol 21:561–569.
Zweifel R, Item H, Häsler R (2000) Stem radius changes and their
relation to stored water in stems of young Norway spruce trees.
Trees 15:50–57.
Zweifel R, Item H, Häsler R (2001) Link between diurnal stem radius
changes and tree water relations. Tree Physiol 21:869–877.
Tree Physiology Online at http://www.treephys.oxfordjournals.org