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J Sci Food Agric 1998, 78, 349È358
Quantitative NMR Imaging of Kiwifruit (Actinidia
deliciosa) during Growth and Ripening
Christopher J Clark,1* Lynley N Drummond2 and Janet S MacFall3
1 HortResearch, Palmerston North Research Centre, Private Bag 11 030, Palmerston North, New Zealand
2 Department of Food Technology, Massey University, Private Bag 11 222, Palmerston North, New
Zealand
3 Department of Biology, Elon College, PO Box 2225, Elon College, North Carolina 27244, USA
(Received 16 May 1997 ; revised version received 9 February 1998 ; accepted 11 March 1998)
Abstract : Quantitative 1H magnetic resonance (MR) imaging was used to determine relaxation changes (T , T -CPMG) at regular intervals during growth and
1 2deliciosa var deliciosa). Temporal trends and difripening of kiwifruit (Actinidia
ferences between Ñesh, locule and core tissue were found for both relaxation
parameters. However, no consistent associations were found between nondestructive measurements and those for individual free sugars, soluble solids
content (SSC) and macronutrients and micronutrients determined on dissected
companion samples. Increases of 200% in total free sugar concentration in Ñesh
and 68% in SSC accompanied starch hydrolysis after harvest. Despite the magnitude of these changes, relaxation times remained unaltered. These observations
were repeated in a second investigation using A arguta fruit and T , T , T 2 2a
CPMG and self-di†usion image contrasts. Here, SSC increased 125%1 during
compressed 15-day ripening period, while MR parameters like self-di†usion
declined only 7È14% from harvest values. T -CPMG relaxation was also investi2
gated in aqueous solutions containing individual
organic acids, sugars or pectate
and juice from ripening fruit (4É7È15É5% SSC). Analysis of solutions and juices
showed relaxation is indeed sensitive to increases in sugar composition but relatively insensitive to changes in organic acids and soluble pectin at concentrations
normally found in fruit. Results imply that relaxation parameters determined
from MR images may not be appreciably inÑuenced by processes that cause
solution composition to vary dramatically, even though these changes are reÑected in the relaxation properties of the juice itself. Possible reasons for this are
discussed with regard to the impact of cell structure and magnetic Ðeld strength
on relaxation processes. ( 1998 Society of Chemical Industry.
J Sci Food Agric 78, 349È358 (1998)
Key words : Actinidia deliciosa ; fruit ; kiwifruit ; magnetic resonance imaging ;
ripening.
INTRODUCTION
detailed representations of vascular architecture
extracted from three dimensional data arrays (McFall
and Johnson 1994). Visual inspection of morphology
is typical of the way in which MRI is used to gather
qualitative information from samples. Obtaining quantitative data concerning the underlying MR parameters
that provide contrast in these images, on the other
hand, requires a di†erent approach. This is achieved
most successfully using quantitative MRI.
Quantitative imaging is an approach whereby images
are constructed that contain the spatial distribution of
Since the seminal study on the use of proton magnetic
resonance imaging (1H MRI) to investigate watercore in
apple (Wang et al 1988), application of MRI to horticultural produce has evolved from simplistic descriptions
of images of samples containing quality disorders, to
* To whom correspondence should be addressed.
Contract/grant sponsor : NZ Foundation for Research,
Science and Technology. Contract/grant number : C06504
349
( 1998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50.
Printed in Great Britain
C J Clark, L N Drummond, J S MacFall
350
static and dynamic characteristics of aqueous protons,
such as their relaxation and di†usive properties. Signal
intensity (S) in a standard NMR experiment depends on
a number of factors :
S \ N [1 [ 2 expM[(T [ T /2)/T N
H
R
E
1
] exp([T /T )] ] exp([T /T )
(1)
R 1
E 2
Here, T (ms), the time between the 90¡ radiofrequency
E
pulse and formation of the spin-echo signal, and T
R
(ms), the recovery time between pulse train repetitions,
are two instrumental parameters, and N , T (ms) and
H 1
T (ms) are three sample-dependent parameters describ2
ing the proton spin-density, the spin-lattice relaxation
time and the spin-spin relaxation time, respectively
(Callaghan 1991). SimpliÐcation of this expression by
choosing appropriate values of T (or T ), and analysing
R
E
the variation in signal intensity as the other instrument
parameter is varied incrementally, leads to the formation of “calculatedÏ images containing relaxation or
proton spin-density data (MacFall JR et al 1987).
Similar procedures enable images containing selfdi†usion and Ñow data to be constructed (Callaghan et
al 1994 ; Kuchenbrod et al 1995, 1996). Evidence suggests that MR parameters reÑect physiological changes
in fruit during ripening or storage (Callaghan et al
1994 ; Ishida et al 1994 ; Clark and MacFall 1996 ;
Goodman et al 1996). To date, there has been no concerted attempt to link changes in MR parameters
obtained from quantitative images to variation in other
physicochemical properties, like carbohydrate composition, which would be expected to a†ect the magnetic
characteristics of cell solutions. Di†erence in soluble
solids content (SSC), for example, is currently being
explored as the basis for an MR sensor to assess sweetness of fruit (Zion et al 1995).
To further understanding of how MR parameters
vary in biological systems, and to determine whether
these might be a useful non-destructive sensor of
physiological processes in fruit, investigations where
quantitative imaging was used to monitor changes in
MR measurements in the fruit and juice of kiwifruit
(Actinidia deliciosa) during growth and ripening are
reported. Additional experiments were also carried out
on an A arguta fruit, a small-fruited relative of kiwifruit
where the ripening period is compressed, and binary
aqueous solutions containing compounds commonly
found in fruit juices. Reasons for selecting Actinidia as a
model exploratory system were twofold : Ðrstly, both
species accumulate large amounts of granular starch
during development, then hydrolyse it extensively to
soluble free sugars following harvest (MacRae et al
1992) ; secondly, a limited preliminary study imaging a
surrogate, a small hermaphrodite male kiwifruit, indicated that signiÐcant changes in MR properties and
strong associations with compositional measurements
might be anticipated (Callaghan et al 1994).
EXPERIMENTAL
Samples
Commercial fruit
Fruit were obtained from 11-year-old vines (cv
Hayward) grown on a T-bar trellis system in a research
orchard near Hamilton (North Island, New Zealand).
Commencing 13 December 1993, approximately 2
weeks after full bloom, between 45 and 160 fruit were
harvested regularly throughout the growing season. At
commercial harvest (9 May 1994 ; 6É2% SSC), twice as
many fruit were sampled. One half was analysed immediately, while the other was allowed to ripen (2 weeks
storage at 4¡C followed by ethylene-induced ripening
for 2É5 weeks at 20¡C). At each sampling date, fruit were
sliced laterally to divide the length of the stem axis into
three equal parts. These basal, median and distal sections were further dissected into the skin plus outer
pericarp (Ñesh), the inner pericarp (locules plus seeds)
and core. Depending on stage of development, segments
from 13 to 45 fruit were bulked to provide sufficient
mass for analysis. This procedure was repeated to give
three replicates of each sample type at every harvest
data. Fresh-weight samples were subsequently weighed,
frozen, lyophilised, reweighed and ground to a powder.
Small fruit
Fruit from an A arguta selection (J5I3) (\2É5 cm
diameter) were harvested on 18 March 1996. To
prolong storage and prevent shrivelling, the mature fruit
were sealed in small plastic containers. SSC (Atago
refractometer) was determined 1, 4, 7, 10, 15 and 21
days after harvest to coincide with imaging, and three
replicates consisting of four fruit each were retained for
compositional analysis. Whole-fruit were processed for
analysis in the same way as larger fruit. However,
because of limited fruit numbers and their small size,
individual tissues were not dissected in this experiment.
Juice and solutions
At weekly intervals prior to commercial harvest, juice
was squeezed from 10-mm segments cut from the top
and bottom of individual fruit (cv Hayward). After commercial harvest, changes were monitored over 4 months
using fruit held in storage (0¡C). On each occasion, juice
from 10 fruit was bulked to provide sufficient solution
(10 ml) for spectroscopic analysis. Solutions were frozen
at [ 80¡C until required. Samples were subsequently
centrifuged prior to use to remove chlorophyll and
denatured protein.
Solutions containing soluble compounds typically
found in fruits were prepared in distilled water. Concentration ranges were decided on the basis of a survey of
compositional analyses of a number of fruits and at different stages during their development. Thus, the ranges
chosen reÑect upper limits encountered in a sample
NMR imaging of kiwifruit
spectrum, not just those in kiwifruit or at harvest.
Materials and concentration ranges used included the
following : sugarsÈglucose, fructose and sucrose, 5, 10
and 20% (w/v) ; polyolsÈinositol 5 and 10% (w/v) and
sorbitol 5, 10 and 20% (w/v) ; organic acidsÈmalic 1
and 2É5% (w/v), citric and quinic 2É5 and 5% (w/v) ; and
pectinÈNa CO -soluble kiwifruit pectin 0É5 and 2É0%
2
3
(w/v). Pectin was kindly supplied by Robert Redgwell.
Details of the extraction and composition of this
material are in Redgwell et al (1992).
Analytical methods
Measurements on freeze-dried powders included the following : determination of macronutrients and micronutrients in nitric/perchloric-acid digests by standard
atomic absorption spectroscopy ; starchÈcolorimetric
detection of glucose after hydrolysis with amyloglucosidase (Smith et al 1992) ; and individual sugars after
extraction and derivatisation for analysis by GC
(Bieleski 1994).
Imaging techniques
Commercial fruit
Proton NMR experiments on commercial fruit were
performed in a 2 T horizontal widebore magnet at
Duke University, NC, USA. Four fruit were imaged at
each harvest date. These were despatched by international courier within 2 h after picking. Because of the
length of time in transit, however, NMR experiments
did not begin until 1 week later and were generally
completed within 2 or 3 days thereafter.
Three small glass capillary tubes containing CuSO
4
internal standards were taped along the top and along
the side of the fruit, so that they would appear in images
of the median longitudinal and transverse planes. Fruit
were positioned in a 10-cm-diameter “birdcageÏ coil with
the long axis of the fruit parallel to the direction of the
bore.
At every imaging session, 28 single-slice, spin-echo
images were obtained from each fruit. Six images using
a standard spin-echo spin warp pulse sequence were
sequentially acquired through each transverse and longitudinal slice plane for calculation of T values (echo
1
time (T ) \ 10 ms ; repetition time (T ) ranging from 100
E
R
to 3200 ms). A further eight images were acquired
through each slice plane for calculation of T values
2
using a CarrÈPurcellÈMeiboomÈGill (CPMG) pulse
sequence (T \ 20È160 ms in 20-ms increments ; T \
E
R
3200 ms). Individual image slices were all 2-mm thick
with an in-plane resolution of between 0É12 and
0É33 mm (256 ] 256 data arrays)Èresolution decreased
as fruit became progressively larger.
Relaxation times were obtained from the “calculatedÏ
images arising from the Ðtting procedures of MacFall
351
et al (1987). Measurements in the transverse plane were
from circular regions of interest (ROIs) randomly selected within the Ñesh, locules (but not seeds) and core.
Those in the longitudinal plane consisted of ROIs from
fresh in basal, median and distal regions of the fruit.
Relaxation measurements for a particular tissue were
calculated as the mean of six ROI measurements, each
in turn the average of relaxation data accumulated over
137 (Ñesh), 21 (locule) or 80 (core) pixels. All measurements were normalised according to the T and T
1
2
values calculated for two of the reference tubes associated with each image, to remove any likelihood of
instrument variability over the course of the study.
Small fruit
A single fruit was selected for the 3-week experiment
involving A arguta. At each imaging session, MR
experiments utilising a 7É1 T 300 MHz Bruker
AMX-300 NMR spectrometer were carried out to
produce a range of image contrasts. These included
standard spin-echo spin warp (T and T -SE), a T 1
2
2
CPMG sequence, gradient echo (T *), and di†usion con2
trast. The pulse sequences and instrument parameters
used are described in Callaghan et al (1994), as are the
techniques employed subsequently to produce the “calculatedÏ images containing the quantitative data for
each contrast. Images, obtained from 1-mm-thick slices,
were all reconstructed and displayed on a 256 ] 256
pixel array giving an in-plane resolution of 98 lm.
Juice and solutions
Transverse relaxation was determined as a function of
pulse-width spacing (PWS) in a CPMG pulse sequence
following the method of Hills and Duce (1990). Data
were acquired using the 300-MHz AMX spectrometer
at Ðxed 90È180¡ pulse spacings varying between 44 ls
and 20 ms. The T time associated with each PWS was
2
determined from linear regression by modelling the
logarithmic transform of that region of the data on the
free induction decay curve associated with aqueous
protons. All measurements were on sample volumes of
about 1 ml at 25¡C.
Transverse relaxation data are presented as the
reciprocal of transverse relaxation (or relaxation rate) vs
the reciprocal of PWS (see Figs 6 and 7). In this form
the dispersion data are able to be modelled using the
two-site chemical exchange model of Carver and
Richards (1972), where the shape of the relationship is
expressed as a function of the concentration of
exchangeable solute protons, amongst other factors.
RESULTS
Commercial fruit (A deliciosa)
Variation in the intensity of individual tissues in calculated images (Figs 1 and 2) indicated the existence of
352
C J Clark, L N Drummond, J S MacFall
Fig 1. Calculated spin-lattice relaxation (T ) images through a longitudinal equatorial plane of kiwifruit (A deliciosa) during
1
development and ripening. Numbers correspond to weeks from fruitset, with commercial harvest occurring at week 23. Image
resolution varies from 0É12 in the youngest fruit to 0É33 mm in the older samples (256 ] 256 pixel data arrays). The circular
internal standard tubes (4 mm inner diameter) serve as a scale marker for each panel. Image intensity is based on a continuous
grey-scale setting ranging from 0 ms \ black to 2500 ms \ white. Note, images at 23 and 27É5 weeks are of the same fruit at
harvest and after 4É5 weeks ripening.
distinct di†erences between the relaxation properties of
the Ñesh, locule and core tissues at particular sampling
dates, as well as trends during the season. Digital information extracted from discrete ROIs from images such
as these formed the basis for quantitative analysis of
these di†erences and trends.
The shortest relaxation times (either T or T -CPMG)
1
2
were those measured in the core (Fig 3). T times for
1
Ñesh and locule tissue increased during fruit development and were of a similar magnitude at individual
harvest dates. In contrast, T -CPMG times di†ered
2
widely among the three tissues from around 12 weeks
after fruitset, proving this to be a more sensitive indicator of changes in magnetic environment than T .
1
There were no di†erences of any consequence
amongst the relaxation properties in basal, median and
distal sections of the fruit. Trends at either extremity
adhered closely to those outlined for the median slice in
Fig 3. Thus, while gradients in nutrient composition
between the top and bottom of the fruit exist (Clark and
Fig 2. Calculated spin-spin relaxation (T -CPMG) images through a transverse equatorial plane of kiwifruit (A deliciosa) during
2
development and ripening. Individual images
are from the same fruit depicted in Fig 1. Numbers, resolution and scale markers are
the same as in Fig 1. Image intensity is based on a continuous grey-scale setting ranging from 0 ms \ black to 350 ms \ white.
NMR imaging of kiwifruit
353
Fig 3. Seasonal variation of (A) the calculated T relaxation
time, (B) the calculated T -CPMG relaxation time1and (C) the
distribution of total free2sugar concentration in Ñesh, locule
and core tissue from a median transverse slice of kiwifruit (A
deliciosa). In (A) and (B), error bars are an LSD based on a
pooled estimate of variance (P \ 0É05) for between-tissue comparisons at any imaging date, and data points are the mean of
six ROI measurements on each of four fruit. In (C), error bars
are LSDs (P \ 0É05) for between-tissue comparisons at speciÐc times, and data points are the mean of three replicates.
Arrows indicate commencement of commercial harvest.
Smith 1988), they appear to have no signiÐcant inÑuence on the magnitude of relaxation properties.
In fact, no consistent, strong associations between
composition and relaxation times were established for
any tissue components. Where strong associations were
indicated, these tended to be dependent on the inÑuence
of a single data point and were discounted. T relax1
ation time in the basal, median or distal regions of Ñesh,
for example, was found to be negatively correlated
(P \ 0É05) with moisture content (%) and the concentrations of P, Ca, Cu or Fe. However, the same relationships were absent for locule and core tissue. Other
correlations were found between measurements in either
a particular tissue or at particular times. T and T 1
2
CPMG were correlated with Ñesh N concentration in
median sections, but not others, while T was negatively
1
correlated with total free sugars at weeks 8 and 23 only.
Many of these associations appear coincidental and
make no obvious physiological sense. Further analysis
using stepwise regression was precluded in that measurements were only recorded on six occasions during
the season.
Dramatic changes in relaxation times following
harvest were entirely absent (Fig 3, Table 1). Free and
total sugars increased between 100 and 300% after
harvest and soluble solids by 68%, as expected (Fig 3,
Table 1). However, this marked alteration in solution
composition was not reÑected in detectable changes in
relaxation properties.
Small fruit (A arguta)
Visual inspection of images from the 21-day A arguta
ripening experiment implied that changes in relaxation
times were minimal. Examples of calculated images produced from each pulse sequence experiment are shown
for day 4 data only (Fig 4). The images are notable for
the lack of contrast between locule and Ñesh (cf Fig 2).
The darker intensity of the core suggests a longer T
1
relaxation time or a shorter transverse relaxation time
relative to the other two tissues. In the case of the selfdi†usion coefficient (D), the darker intensities are consistent with increased solution viscosity or slower
di†usion. With the exception of the seeds and core, the
TABLE 1
Relationship between maturation status, type of tissue and magnetic and compositional properties of unripe kiwifruit (A
deliciosa) at commercial harvest (6É2% soluble solids content) and at eating ripeness 1 month latera
Parameter
T
1
T -CPMG
2
Fructose
Glucose
Sucrose
Total soluble sugars
Total soluble solidsb (%)
Flesh
L ocule
Core
Unripe
Ripe
P
Unripe
Ripe
P
Unripe
Ripe
P
1610
201É4
13É8
12É0
5É4
32É4
6É3
1809
215É9
45É7
41É8
16É7
106É9
10É6
NS
NS
**
NS
**
*
***
1774
344É3
10É9
10É1
5É7
28É0
1792
354É6
34É3
32É0
11É0
79É6
NS
NS
**
***
*
**
874
88É9
14É2
12É3
7É6
37É9
1370
105É8
49É2
40É4
29É2
126É1
NS
NS
***
***
*
***
a Relaxation data were collected from the same four fruit on each occasion. Probability (P) is based on a t-test with n \ 6
degrees of freedom for relaxation measurements and n \ 4 for sugars. Units are ms for relaxation and mg g~1 dry weight
for carbohydrates.
b Whole fruit measurement.
354
C J Clark, L N Drummond, J S MacFall
Fig 4. Tissue contrasts in A arguta derived from di†erent pulse sequence experiments. Calculated images show spin-lattice relaxation (T , 0È2500 ms), spin-spin relaxation (T se, 0È200 ms), gradient echo (T *, 0È4 ms), spin-spin relaxation using a CPMG pulse
2
2 obtained from the T -CPMG sequence, and the
sequence1 (T cp, 0È300 ms), pseudo proton spin-density
(0È2000 arbitrary units)
2
2
self-di†usion coefficient for water (0È3É5 ] 10~9 m2 s~1), respectively. Ranges in parentheses refer to values delineating the greyscale setting in each panel. All images are of the same fruit at 4 days after harvest and are based on 256 ] 256 pixel data arrays
(resolution 98 lm), except the self-di†usion image which is 128 ] 128. The scale marker (5 mm) in the Ðrst image applies to each
panel.
pseudo proton spin-density map (Fig 4) indicates that
the distribution of water is uniform throughout the
inner and outer pericarp. Relaxation times in the
gradient-echo (T *) experiments proved too short
2
(\5 ms) to be adequately measured.
Examination of quantitative measurements conÐrmed
that changes in relaxation were small, and, where there
were temporal trends, these were gradual rather than
abrupt (Fig 5). SpinÈspin relaxation (T -SE) proved to
2
be the most sensitive technique for discriminating
between various tissues. As with the larger fruit, core
tissue had the shortest relaxation time relative to the
other tissues, and the slowest D coefficient (Fig 5).
Measurements were compared 1 day after harvest
and after 15 days ripening when the fruit were eatingripe (by day 21 fruit were too soft and over-ripe) (Table
2). As expected, the SSC of companion A arguta fruit
increased sharply from 8É2 to 18É6% (P \ 0É001) during
ripening. In contrast to this 125% increase in soluble
solids, variation in the MR parameters was small : T
1
increased by 11%, but only in the core ; T -SE decreased
2
by 22È25% in the Ñesh and core ; and T -CPMG
2
decreased by 10% in the core (Table 2). Di†usion was
the single measurement in which all three tissues
demonstrated a consistent e†ect, declining 7È14% from
their harvest values.
Juice and solutions
Fig 5. Variation of (A) the calculated T -SE relaxation time
2
and (B) the self-di†usion coefficient (units ] 1012 m2 s~1) in
Ñesh, locule and core tissue from a median transverse slice of
an A arguta fruit during ripening. Error bars are LSDs for
between-tissue comparisons at individual imaging dates. Data
points are the mean of eight separate ROI measurements.
The appearance of the relaxation curves indicated that
fast proton exchange between water and solute was the
dominant mechanism involved in T relaxation in each
2
of the individual solution studies (Hills et al 1989 ; Hills
and Duce 1990). The salient features have been summarised using a subsample of the experiments carried
out (Fig 6).
Pectates are released from cell walls during the ripening processes (Redgwell et al 1992). While their concentration in solution is not high (typically 0É08È0É5%
(w/v)), they may have a profound e†ect on solution
properties. At a concentration of 0É5% (w/v) and pH
4É2, the relaxation properties of pectate solution
NMR imaging of kiwifruit
355
TABLE 2
Relaxation (ms) and self-di†usion (]1012 m2 s~1) in an A arguta fruit, 1 and 15 days after harvesta
Parameter
T
1
T -SE
2
T -CPMG
2
Self-di†usion
Flesh
L ocule
Core
Day 1
Day 15
P
Day 1
Day 15
P
Day 1
Day 15
P
1339
128É0
141É7
2003
1362
100É4
135É5
1860
NS
**
NS
***
1352
133É9
166É4
1929
1355
139É7
141É9
1656
NS
NS
NS
***
949
106É1
69É3
1465
1050
79É9
62É5
1253
**
***
**
**
a The probability (P) is based on a paired t-test with n \ 7 degrees of freedom.
(D1800 ms) were very similar to that of pure water
(D2000 ms) (Fig 6A). Addition of acid to adjust this
pectate solution to pH 3, or increasing the concentration of pectate itself to 2É0% (w/v), caused formation of
a viscous gel. In both cases, relaxation times declined to
around 680 and 500 ms, respectively. Spin-spin relaxation of water and pectate solutions/gels showed no
dependence on PWS.
Solutions of the weak acids, malic (pK \ 3É4) and
1
quinic (pK \ 3É7), were prepared at pH 4 by neutral1
isation with KOH. Equivalent concentrations (1% w/v)
had di†erent relaxation times (2500 vs 2100 ms) with
relaxation times declining to 2400 ms (2É5% (w/v)
malate) and 1500 ms (5% (w/v) quinate) at the higher
concentrations associated with fruit. In each case, relaxation was independent of PWS. The fact that the two
1% (w/v) solutions do not have the same transverse
Fig 6. SpinÈspin relaxation rates (1/T ) for solutions containing compounds found in fruit juices as2a function of reciprocal
90È180¡ CPMG pulse-width spacing (PWS) : (A) Na CO 3
extractable pectin from kiwifruit ; (B) organic acids ; (C)2 fructose at di†erent concentrations ; (D) fructose, glucose and
sucrose ; (E) e†ect of pH on citric acid ; and (F) e†ect of pH on
sucrose.
relaxation time is not surprising. With four potentially
exchangeable hydroxyl protons on quinate as opposed
to one in malate, on a molar basis, 1% (w/v) quinate
solution has almost three times as many exchangeable
protons as malate. The observed relaxation rate (1/T )
2
would be expected to be faster in the solution with the
greater number of exchangeable solute protons (Carver
and Richards 1972).
In contrast, relaxation properties of sugar solutions
varied depending on the molecule, its concentration and
the PWS selected (Fig 6C, D). For example, relaxation
times in 5, 10 and 20% (w/v) fructose solutions ranged
between 100È2000 ms, 50È1700 ms and 20È1400 ms,
longer relaxation times being associated with shorter
PWS. This pattern was evident for other free sugars,
plus inositol and sorbitol (data not presented). The 20%
fructose solution was unique, however, with respect to
having exceptionally short relaxation times below PWS
of 2É5 ms. In other simple carbohydrate systems, relaxation times were generally stable below 2É5 ms (cf Fig
6D). Samples of equivalent concentration (10% (w/v))
show clearly that relaxation is a†ected by the type of
molecule present, even when they have the same
number of exchangeable protonsÈas with fructose and
glucose (Fig 6D). Here, relaxation times in fructose (50È
1700 ms) were shorter than those in glucose (74È
1700 ms) and sucrose (100È2000 ms).
Acidity also a†ected relaxation rate (Fig 6E, F). An
increase in acidity of a 1% (w/v) citrate solution from
pH 4 to 3, caused relaxation times to decrease from
2400 to 2300 ms (Fig 6E). In the case of sucrose (10%
(w/v)), increasing acidity from pH 5 to 3 changed relaxation times from between 100 and 2000 ms to between
520 and 700 ms, at the same time removing the dependency on PWS (Fig 6F). For most PWS, this represents
an increase in the relaxation time, the opposite of the
trend for citric acid.
The relaxation rate of kiwifruit juice increased with
increasing SSC and showed a marked dependence on
PWS at SSC greater than about 8É4% (Fig 7). Fructose
and glucose are the major components in these juices.
Solution pH (unadjusted) averaged 3É3 and ranged
between 3 and 4. Perseverance of a pulse-width dependence in these juice extracts (despite their high acidity)
C J Clark, L N Drummond, J S MacFall
356
Fig 7. SpinÈspin relaxation rates (1/T ) for kiwifruit juice
2
obtained from fruit at di†erent stages of ripening (soluble
solids content) as a function of reciprocal 90È180¡ CPMG
pulse-width spacing (PWS).
indicates that acid does not inÑuence relaxation is this
more complex system to the same extent as its addition
does in single component systems.
DISCUSSION
The results of quantitative MR imaging during growth
and ripening of kiwifruit indicate that there are distinct
tissue and seasonal di†erences in the relaxation measurements, but the measurements do not necessarily
reÑect
underlying
biochemical
processes.
In
commercial-sized fruit, it is quite clear that the relaxation parameters determined from quantitative images
are insensitive to major physiological events that have a
dramatic impact on solution composition. Furthermore,
the lack of change in the relaxation measurements postharvest cannot be attributed to compounds present in
the juice itself. The observation that shorter relaxation
times are associated with increasing SSC in juices, or
with increasing concentration in binary aqueous solutions, is consistent with the Carver and Richards (1972)
formulation, in which T is inversely related to the con2
centration of exchangeable solute protons, and experimental results relating relaxation and carbohydrate
concentration (Mora-Gutierrez and Baianu 1989 ; Birch
and Karim 1992). Failure to obtain these results via the
imaging process indicates that other factors must be
involved.
Two factors that might contribute to insensitivity of
the imaging data to changes in solution composition are
choice of PWS and magnetic Ðeld strength. The sugar
solution studies indicate that relaxation becomes less
dependent on concentration at PWS \ 100 ls. This can
be discounted here, however, since the T -CPMG
2
imaging experiments utilised a PWS of 2É5 ms (1/
PWS \ 400 s~1). This is where di†erences between individual sugars or change in concentration would be
expected to have a substantial impact on T relaxation
2
(Fig 6D). T dispersion e†ects also originate from
2
exchange of protons between sites with di†erent chemical shift values (eg sugar hydroxyl protons vs aqueous
protons). The absolute value of the chemical shift di†erence between such sites is, in turn, strongly inÑuenced
by the choice of magnetic Ðeld strength. Thus, di†erences relating to solution composition observed at
7É1 T (Fig 6) will be much less at 2 T, the Ðeld strength
at which commercial fruit were imaged. This may
account for lack of observation of signiÐcant T changes
2
in commercial-sized fruit postharvest. It does not
account for the fact that the authors failed to observe T
2
changes during ripening of the small A arguta fruit,
which was imaged at the same Ðeld strength as the
binary solutions.
Other possibilities relate to modulation of relaxation
by a sampleÏs structural features. Intercellular air
spaces, for example, produce localised magnetic Ðeld
gradients at solutionÈair interfaces. With a pulse
sequence like T -SE, di†usion of water molecules
2
through these gradients enhances the rate of relaxation
and may produce susceptibility artefacts in the image
itself (Callaghan 1991). Transverse relaxation in parenchyma of courgette, onion and apple have been
described using combinations of fast proton exchange
between water and solutes and di†usion through the
internally generated Ðeld gradients at cellÈair interfaces
(Hills and Duce 1990). Di†usion between intracellular
and intercellular aqueous-Ðlled compartments also
averages signal intensity. Hills and Duce (1990) combined these factors in a two-compartment cellular relaxation model based on chemical and di†usive exchange.
Semi-quantitative Ðtting of their fruit and vegetable
data indicated that cell morphology and membrane permeability strongly inÑuenced T relaxation. Links
2
between the multi-exponential nature of transverse
relaxation in biological cells and cell geometry have
also been proposed by Brownstein and Tarr (1979).
From their model, based solely on the properties of
bulk water and characterisation of the cell wall as a
surface sink, it is possible to infer estimates of both the
shape and size of the cells from the NMR data. Detailed
information concerning the size, shape, number and
ultra-structure of kiwifruit cells during growth and
ripening have been documented (Hopping 1976 ; Hallett
et al 1992). Further experiments with large fruit at both
low and high Ðeld strengths, and modelling the pulsewidth dependence of transverse relaxation data from
di†erent tissues during development and ripening (Hills
and Duce 1990), is an obvious path forward in order to
probe the compositional and structural ramiÐcations
outlined here.
Nevertheless, where changes in relaxation measurements are observed by MRI which are likely to be
related to speciÐc physiological events (Callaghan et al
1994 ; Clark and MacFall 1996), the solution studies
contain information relevant to their interpretation. For
instance, organic acids at concentrations commonly
found in fruits, and kiwifruit pectate solution, have long
relaxation times similar to bulk water (Fig 6A, B).
Hence, concentration changes involving these compounds should have little e†ect on MR data, except in
NMR imaging of kiwifruit
circumstances where change in pH leads to acid catalysis of proton exchange between carbohydrates and
water (the present study ; Hills 1991) or gelation of
pectin. The concentrations of individual free sugars, on
the other hand, vary widely during the season. Unfortunately, in addition to their dependence on PWS and
concentration, solutions containing the same number of
exchangeable protons (fructose and glucose) do not
have equivalent relaxation properties either (Fig 6D). At
extremely high concentrations ([100% (w/v)) these
variations are attributed to di†erences in viscosity or
solubility (Suggett et al 1976 ; Lai and Schmidt 1991). At
more dilute concentrations however, where viscosity differences between solutions of fruit sugars are small,
variations in relaxation probably relate to conformation
changes in solution a†ecting the ratio of axial/
equatorial hydroxyl protons and ease of exchange with
aqueous protons (Harvey and Symons 1978 ; MoraGutierrez and Baianu 1989). Moreover, in ternary
aqueous systems containing simple sugars plus neutral
salts, relaxation rates are inÑuenced by ionic strength
and the composition of the neutral salt (Bociek and
Franks 1979 ; Lai and Schmidt 1991 ; Sattiacoumar and
Arulmozhi 1993). Setting aside any e†ects structure may
impose on relaxation, the array of factors capable of
inÑuencing relaxation in sugarÈwater solutions serves to
emphasise that variations in serial imaging data can not
be interpreted unequivocally without recourse to
detailed compositional analysis. Localised spectroscopy
and chemical shift imaging can, of course, be used to
probe carbohydrates directly in imaging experiments
rather than trying to infer their presence based on
changes in relaxation alone (Ishida et al 1994, 1996 ; Tse
et al 1996).
The failure of imaging to detect large changes in solution composition is not inconsistent with attempts to
develop other NMR techniques as non-destructive
sensors for sweetness in fruit. Interference from localised
Ðeld gradients in two-dimensional data collection is
avoided by analysing the spectral characteristics of the
whole fruit. Use of surface coils to generate homogeneous magnetic Ðelds in particular regions of the sample
and analysing the sugar/water ratio of their respective
spectral peaks (Zion et al 1995), or using the strong
relationship between self-di†usion and sucrose concentration in aqueous solution (Wai et al 1995), are the
more successful approaches currently being evaluated.
What is unusual about the small male fruit used
in our exploratory investigation where relaxation
changes at 7É1 T were observed after harvest (Callaghan
et al 1994) ? The only obvious di†erence is that the concentration of free sugars in the small ripened fruit
exceeded 400 mg g~1 dry weight. This is at least four
times greater than that recorded in commercial fruit.
Perhaps thresholds as high as this have to be exceeded
before solution e†ects overcome those imposed by
structural constraints ? However, even at concentrations
357
as high as this, the self-di†usion coefficient in male fruit
declined by only 12% compared with the 28% that
might otherwise be expected from the relationship
between self-di†usion and SSC in sucrose solutions
(Irani and Adamson 1958). Hence, even under circumstances where the greatest possible changes in solution
composition occur, changes in MRI measurements are
not especially sensitive.
The results indicate that quantitative MRI is able to
measure temporal trends and tissue di†erences in MR
properties of kiwifruit. One can attempt to rationalise
these on the basis of changes in solution composition.
The tendency towards longer relaxation times later in
the season, in locular tissue for instance, is certainly
consistent with increasing dilution of cell contents, as
dominant soluble macronutrients like N and K become
less concentrated during fruit development (Clark and
Smith 1988). However, this ignores other factors that
have an impact on relaxation, like increase in cell size
(Hopping 1978) and change in the volume of the intercellular gas space postharvest (Hallett et al 1992).
Hence, interpretation of data from quantitative images
of biological samples will require a more intimate, holistic view of what is occurring within the sample, and at a
level of detail which will be difficult to provide for many
of the systems that are currently of interest. Indeed, the
results suggest that fruit systems are probably too
complex for even a basic set of relaxation principles to
apply across a range of crops.
ACKNOWLEDGEMENTS
The authors thank Rod Bieleski, Ken Marsh and
Vivienne Paterson for technical assistance ; Barbara
Dow for support with statistical analysis : Paul Callaghan for pulse-sequence programming ; and Allan Johnson
for generous access to Duke University Radiology
Department facilities. This study was funded by the NZ
Foundation for Research, Science and Technology
(C06504), and Ðnancial support from the NZ Kiwifruit
Marketing Board is acknowledged.
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