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Identification of the distribution of sugars in grapes using chemical shift selective NMR microscopy
Magneric Resonsnce Imaging, Vol. II, pp. 1039-1041, Printed in the USA. All rights reserved.
1993 Copyright 0
0730-725X/93 $6.00 + .CKl 1993 Pergamon Press Ltd.
l Technical Note
IDENTIFICATION OF THE DISTRIBUTION USING CHEMICAL SHIFT SELECTIVE B.A. GOODMAN,*
B. WILLIAMSON,*
OF SUGARS IN GRAPES NMR MICROSCOPY
AND J.A.
CHUDEK?
*Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK and tDepartment of Chemistry, University of Dundee, Dundee DDl 4HN, Scotland, UK Images are presented which show that NMR microscopy can be used to determine the spatial distribution of sugars in grapes. The production of appreciable sugar gradients during the final ripening process was detected in parenchymatous tissues around the seeds, but these gradients disappeared in fully ripened fruit. Keywords: Grape; Sugar; NMR microscopy; Chemical shift selective imaging.
essary to excite only the frequency of interest in the NMR spectrum. In our measurements the length of a Gaussian soft pulse, attenuated to 90”, was fixed to the inverse of the width of the resonance peak of interest; both transmitter and receiver frequencies were that of the resonance. Images from a ripening grape of the water and sugar components, which correspond to peaks in the NMR spectrum at 4.6 and 3.4 ppm, respectively, are shown in Fig. la and b. The sugar image shows a pronounced concentration gradient radiating from the seeds. This is in contrast to the image of Pope et aL3 which consisted of a sharply defined halo, which essentially followed the outline of the seed as seen in the water image. We believe that the image in Fig. lb is not consistent with an artifact induced by magnetic susceptibility variations in the surface layers of the seeds. Furthermore, an image of the same slice (Fig. lc) obtained with a gradient-echo sequence, which is well-known to be sensitive to magnetic susceptibility variations in biological specimens,” showed no features that corresponded with the boundaries of the regions of enhanced intensity in Fig. lb. Improved resolution of structural details within the parenchymal tissue was observed, however, using the gradient echo imaging technique (Fig. lc compared with Fig. la), presumably as a result of local magnetic susceptibility variations. We conclude, therefore, that the image in Fig. lb shows a genuine sugar gradient in the specimen. It is interesting to note that we did not see such gradients
Elevated levels of sugars close to seed surfaces have been reported for developing fruits of grape and have been attributed to local elevated temperatures of tissues associated with the body of the seed.‘,’ Pope et a1.3 have suggested that the spatial distributions of sugars could be seen by chemical shift selective NMR imaging and presented images from ripe and overripe grapes in which there was considerable enhancement of the intensity of the signal centered on 6 = 3.6 ppm in the region of the seed surface. This is the position expected for the sugars gIucose and fructose, but the shape of the image was surprising, in that it appeared to be associated more with the outer parts of the seed than with the parenchyma tissues of the fruit. It has now been suggested4 that this image is an artifact produced by the presence of air pockets in some of the seed traces. As part of our work on evaluation of the application of NMR microscopy to the characterisation of fruit and the elucidation of developmental processes,5-‘0 we have also performed measurements using a variety of imaging procedures on berries of grape, cv. Black Hambourg, a hardy cultivar suitable for culture in Scotland under glass without supplementary heating, but otherwise having no unusual characteristics. Among these, we have observed chemical shift selective images of grapes at the “ripening” stage when purple pigmentation develops, in which we believe there is resolution of the concentration gradient of sugars that was originally hypothesised by Pope et a1.3 In order to achieve chemical shift selectivity, it is nec-
Crop Research Institute, Scotland, UK.
RECEIVED 10/23/92; ACCEPTED S/3/93. Address correspondence to Dr. B.A. Goodman, Scottish 1039
Invergowrie,
Dundee DD2 SDA,
Magnetic Resonance Imaging 0 Volume I I, Number 7, 1993
1040
Fig. 1. ‘H NMR microscopic images at 7 T with the imaging plane perpendicular to B, of grapes (Black Hambourg). (a) Water image (resonance at 4.6 ppm) of ripening fruit obtained using the Bruker standard XYSIMAGE sequence with the following parameters: Relaxation delay = 1 s; shift selective pulse (Gaussian shape) = 3.5 ms; slice selective pulse (Hermitian shape) = 4 ms; echo time = 18.85 ms; and acquisition time = 10.24 ms. (b) Sugar image (resonance at 3.4 ppm) obtained using the same sequence and parameters as (a). (c) Image of the same slice as that in (a) and (b) obtained using the Bruker gradient-echo sequence XYFAST and the following parameters: Relaxation delay = 0.1 s; excitation pulse (Hermitian shape) = 4 ms attenuated to give a 10” pulse; echo time = 12.24 ms; and acquisition time = 10.24 ms. (d) Sugar image of an unripe grape obtained using the same experimental parameters as (b). The gradients for all images were set to give voxel dimensions of 100 pm3. The poorer signal-to-noise in (d) compared with that in (b) is the consequence of the low sugar contents in the unripe grape.
in “fully
ripe”
or “unripe”
specimens.
Our
chemical
shift selective images of these specimens (Fig. Id and unpublished results) showed an essentially uniform distribution of sugar throughout the tissue, as has also been
reported
by Pope
4.
et a1.4
Acknow/edgments-Financial support for this work was provided by the Scottish Office Agriculture and Fisheries Department. We are also grateful to Dr. D.J.F. Brown for the donation of the grape
5.
specimens.
6.
REFERENCES Possner, D.R.E.; Kliewer, W.M. The localisation of acids, sugars, potassium and calcium in developing grape berries. Vitis 24:229-240; 1985. Coombe, B.G. Distribution of solutes in the developing grape berry in relation to its morphology. Am. .I Enol. Vitic. 38:120-127; 1987. Pope, J.M.; Rumpel, H.; Kuhn, W.; Walker, R.; Leach,
7.
8.
D.; Sarafis, V. Applications of chemical shift selective NMR microscopy to the noninvasive histochemistry of plant materials. Magn. Reson. Imaging 9:357-363; 1991. Pope, J.M.; Walker, R.R.; Kron, T. Artifacts in chemical shift selective imaging. Mugn. Reson. Imaging 10: 695-698; 1992. WiIliamson, B.; Goodman, B.A.; Chudek, J.A. Nuclear magnetic resonance (NMR) micro-imaging of ripening red raspberry fruits. New Phytol. 120:21-28; 1992. Goodman, B.A.; Williamson, B.; Chudek, J.A. Nuclear magnetic resonance (NMR) microimaging of raspberry fruit: Further studies on the origin of the image. New Phytol. 122:529-535; 1992. Goodman, B.A.; Williamson, B.; Chudek, J.A. Noninvasive observation of the development of fungal infection in fruit. Protoplusma 166:107-109; 1992. Williamson, B.; Goodman, B.A.; Chudek, J.A.; Johnston, D. J. Nuclear magnetic resonance (NMR) microimaging of soft fruits infected by Rofrytis cinerea. In: K.
Sugar distribution in grapes 0 B.A. GOODMAN ET AL. Verhoeff, N.E. Malathrakis, B. Williamson (Eds). Recent advances in Botrytis research. Proceedings of the 10th International Botrytis Symposium, Heraklion, Crete. Wageningen: Pudoc Scientific Publishers; 1992: 140-144. 9. Brennan, R.; Goodman, B.A.; Chudek, J.A. Freezing events in flowers of Ribes nigrum L. revealed by NMR microimaging. .I. Hort. Sci. (accepted for publication).
1041
10. Williamson, B.; Goodman, B.A.; Chudek, J.A. The structure of mature gooseberry (Ribesgrossulariu) fruits revealed non-invasively by NMR microscopic imaging. Micron (accepted for publication). 11. Bowtell,R.W.;Brown,G.D.;Glover,P.M.;McJury,M.; Mansfield, P. Resolution of cellular structures by NMR microscopy at 11.7 T. Phil. Trans. Roy. Sot. London (A) 333:457-467;
1990.
1993 Copyright 0
0730-725X/93 $6.00 + .CKl 1993 Pergamon Press Ltd.
l Technical Note
IDENTIFICATION OF THE DISTRIBUTION USING CHEMICAL SHIFT SELECTIVE B.A. GOODMAN,*
B. WILLIAMSON,*
OF SUGARS IN GRAPES NMR MICROSCOPY
AND J.A.
CHUDEK?
*Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK and tDepartment of Chemistry, University of Dundee, Dundee DDl 4HN, Scotland, UK Images are presented which show that NMR microscopy can be used to determine the spatial distribution of sugars in grapes. The production of appreciable sugar gradients during the final ripening process was detected in parenchymatous tissues around the seeds, but these gradients disappeared in fully ripened fruit. Keywords: Grape; Sugar; NMR microscopy; Chemical shift selective imaging.
essary to excite only the frequency of interest in the NMR spectrum. In our measurements the length of a Gaussian soft pulse, attenuated to 90”, was fixed to the inverse of the width of the resonance peak of interest; both transmitter and receiver frequencies were that of the resonance. Images from a ripening grape of the water and sugar components, which correspond to peaks in the NMR spectrum at 4.6 and 3.4 ppm, respectively, are shown in Fig. la and b. The sugar image shows a pronounced concentration gradient radiating from the seeds. This is in contrast to the image of Pope et aL3 which consisted of a sharply defined halo, which essentially followed the outline of the seed as seen in the water image. We believe that the image in Fig. lb is not consistent with an artifact induced by magnetic susceptibility variations in the surface layers of the seeds. Furthermore, an image of the same slice (Fig. lc) obtained with a gradient-echo sequence, which is well-known to be sensitive to magnetic susceptibility variations in biological specimens,” showed no features that corresponded with the boundaries of the regions of enhanced intensity in Fig. lb. Improved resolution of structural details within the parenchymal tissue was observed, however, using the gradient echo imaging technique (Fig. lc compared with Fig. la), presumably as a result of local magnetic susceptibility variations. We conclude, therefore, that the image in Fig. lb shows a genuine sugar gradient in the specimen. It is interesting to note that we did not see such gradients
Elevated levels of sugars close to seed surfaces have been reported for developing fruits of grape and have been attributed to local elevated temperatures of tissues associated with the body of the seed.‘,’ Pope et a1.3 have suggested that the spatial distributions of sugars could be seen by chemical shift selective NMR imaging and presented images from ripe and overripe grapes in which there was considerable enhancement of the intensity of the signal centered on 6 = 3.6 ppm in the region of the seed surface. This is the position expected for the sugars gIucose and fructose, but the shape of the image was surprising, in that it appeared to be associated more with the outer parts of the seed than with the parenchyma tissues of the fruit. It has now been suggested4 that this image is an artifact produced by the presence of air pockets in some of the seed traces. As part of our work on evaluation of the application of NMR microscopy to the characterisation of fruit and the elucidation of developmental processes,5-‘0 we have also performed measurements using a variety of imaging procedures on berries of grape, cv. Black Hambourg, a hardy cultivar suitable for culture in Scotland under glass without supplementary heating, but otherwise having no unusual characteristics. Among these, we have observed chemical shift selective images of grapes at the “ripening” stage when purple pigmentation develops, in which we believe there is resolution of the concentration gradient of sugars that was originally hypothesised by Pope et a1.3 In order to achieve chemical shift selectivity, it is nec-
Crop Research Institute, Scotland, UK.
RECEIVED 10/23/92; ACCEPTED S/3/93. Address correspondence to Dr. B.A. Goodman, Scottish 1039
Invergowrie,
Dundee DD2 SDA,
Magnetic Resonance Imaging 0 Volume I I, Number 7, 1993
1040
Fig. 1. ‘H NMR microscopic images at 7 T with the imaging plane perpendicular to B, of grapes (Black Hambourg). (a) Water image (resonance at 4.6 ppm) of ripening fruit obtained using the Bruker standard XYSIMAGE sequence with the following parameters: Relaxation delay = 1 s; shift selective pulse (Gaussian shape) = 3.5 ms; slice selective pulse (Hermitian shape) = 4 ms; echo time = 18.85 ms; and acquisition time = 10.24 ms. (b) Sugar image (resonance at 3.4 ppm) obtained using the same sequence and parameters as (a). (c) Image of the same slice as that in (a) and (b) obtained using the Bruker gradient-echo sequence XYFAST and the following parameters: Relaxation delay = 0.1 s; excitation pulse (Hermitian shape) = 4 ms attenuated to give a 10” pulse; echo time = 12.24 ms; and acquisition time = 10.24 ms. (d) Sugar image of an unripe grape obtained using the same experimental parameters as (b). The gradients for all images were set to give voxel dimensions of 100 pm3. The poorer signal-to-noise in (d) compared with that in (b) is the consequence of the low sugar contents in the unripe grape.
in “fully
ripe”
or “unripe”
specimens.
Our
chemical
shift selective images of these specimens (Fig. Id and unpublished results) showed an essentially uniform distribution of sugar throughout the tissue, as has also been
reported
by Pope
4.
et a1.4
Acknow/edgments-Financial support for this work was provided by the Scottish Office Agriculture and Fisheries Department. We are also grateful to Dr. D.J.F. Brown for the donation of the grape
5.
specimens.
6.
REFERENCES Possner, D.R.E.; Kliewer, W.M. The localisation of acids, sugars, potassium and calcium in developing grape berries. Vitis 24:229-240; 1985. Coombe, B.G. Distribution of solutes in the developing grape berry in relation to its morphology. Am. .I Enol. Vitic. 38:120-127; 1987. Pope, J.M.; Rumpel, H.; Kuhn, W.; Walker, R.; Leach,
7.
8.
D.; Sarafis, V. Applications of chemical shift selective NMR microscopy to the noninvasive histochemistry of plant materials. Magn. Reson. Imaging 9:357-363; 1991. Pope, J.M.; Walker, R.R.; Kron, T. Artifacts in chemical shift selective imaging. Mugn. Reson. Imaging 10: 695-698; 1992. WiIliamson, B.; Goodman, B.A.; Chudek, J.A. Nuclear magnetic resonance (NMR) micro-imaging of ripening red raspberry fruits. New Phytol. 120:21-28; 1992. Goodman, B.A.; Williamson, B.; Chudek, J.A. Nuclear magnetic resonance (NMR) microimaging of raspberry fruit: Further studies on the origin of the image. New Phytol. 122:529-535; 1992. Goodman, B.A.; Williamson, B.; Chudek, J.A. Noninvasive observation of the development of fungal infection in fruit. Protoplusma 166:107-109; 1992. Williamson, B.; Goodman, B.A.; Chudek, J.A.; Johnston, D. J. Nuclear magnetic resonance (NMR) microimaging of soft fruits infected by Rofrytis cinerea. In: K.
Sugar distribution in grapes 0 B.A. GOODMAN ET AL. Verhoeff, N.E. Malathrakis, B. Williamson (Eds). Recent advances in Botrytis research. Proceedings of the 10th International Botrytis Symposium, Heraklion, Crete. Wageningen: Pudoc Scientific Publishers; 1992: 140-144. 9. Brennan, R.; Goodman, B.A.; Chudek, J.A. Freezing events in flowers of Ribes nigrum L. revealed by NMR microimaging. .I. Hort. Sci. (accepted for publication).
1041
10. Williamson, B.; Goodman, B.A.; Chudek, J.A. The structure of mature gooseberry (Ribesgrossulariu) fruits revealed non-invasively by NMR microscopic imaging. Micron (accepted for publication). 11. Bowtell,R.W.;Brown,G.D.;Glover,P.M.;McJury,M.; Mansfield, P. Resolution of cellular structures by NMR microscopy at 11.7 T. Phil. Trans. Roy. Sot. London (A) 333:457-467;
1990.