JOURNAL
OF STRUCTURAL
A Nuclear
BIOLOGY
108,
1%%201
(19%)
Magnetic Resonance Imaging Technique Studies of Water in Plant Leaves DOUGLAS
*Department
lJniuersLt+y Uniuerssty
of Southern Mississippi, of Wisconsin, Madison,
Received April 29, 1991, and in revised form February
A new imaging technique is described which uses nuclear magnetic resonance (NMR) to create a water profile of plant leaves. The water profile shows the average distribution of water as a function of depth into a leaf along a line perpendicular to the leaf surface: it can be used to measure the thickness of cell layers and the quantity of water in each layer. Two-dimensional NMR methods were used to avoid chemical shift distortion which degrades the resolution in leaf images made by conventional NMR techniques; image resolution was improved further by deconvolution analysis. To illustrate its application, the technique was used to follow changes in the internal structure of developing leaves. Q 1992 Academic Press, Inc.
INTRODUCTION
Nuclear magnetic resonance (NMR) techniques can be used to create images that show the distribution of resonating atomic nuclei in a sample. When the sample is living tissue and the instrument is tuned to hydrogen nuclei, the images show predominantly the distribution of liquid water. Medical applications of NMR imaging are well developed (Moonen et al., 19901, and the technique is beginning to be used for botanical research (see, for example, Veres et al., 1991). Raw data from a typical NMR imaging experiment consist of three-dimensional information encoded in a computer memory; the usual practice is to view selected two-dimensional (2-D) slices through the data set. However, NMR imaging is not limited to the three Cartesian axes of geometrical space. Any number of dimensions can be imaged, and the dimensions may include nongeometrical NMR variables. We have developed a 2-D NMR imaging technique in which one dimension represents depth into a layered sample ialong an axis perpendicular to the ’ To whom correspondence
should be addressed.
for
AND JOHN L. MARKLEY?
C. McCAIN*,I
of Chemistry and BiochemLstry, and +Biochemistry Department,
Designed
Hattiesburg, Wisconsin
Mississippa 53706
39406;
1, 1992
layer surfaces), while the other dimension shows chemical shift (a magnetic effect related to the chemical environment of the nuclei) (MeCain et al., 1991); the data are presented as contour plots that show how water in the sample is distributed across the two dimensions. The technique was designed specifically for studies of plant leaves. By including a chemical shift dimension, the method cancels certain distortions that would blur a purely geometrical image, and it produces information that-can be used to distinguish chloroplast water from water that is not located in the chloroplasts. By restricting the number of geometrical dimensions to one, the method focuses on the average distribution of water among cell layers in a planar, layered leaf. We foresee at least five applications of the 2-D imaging technique to studies of plant leaves. The technique can provide (1) images that show the distribution of water as a function of depth into the leaf, (2) separate images of chloroplast water and nonchloroplast water, (3) separate images of vacuolar water and nonvacuolar water, (4) data related to the movement, diffusion or exchange of water between adjacent cells or subcellular compartments in the leaf, and (5) information about the distribution of bound water in a leaf. The objective of this paper is to introduce the new technique. By concentrating on the first application listed above, we describe how water profiles are obtained and discuss how they may be interpreted. We then illustrate the use of water profiles in a study of leaf development. Future papers will discuss the other applications. EXPERIMENTAL Healthy leaves were harvested from the following species: Norway maple (Acer plutunoides L. cv. “Emerald Queen”), lilac, (Syringa uulgaris L. cv. “Madame Lemoine”), cotton (Gossypium hirsutum L. cv. “Texas Marker 1’7, willow (Salti alba L. cv. “Vitellina”), tulip tree (Liriodendron tulipifera L.), and cottonwood Populus deltoides Bartr. ex Marsh). The cotton plant was grown outdoors in a pot; others grew on the campus of the University of
1047-8477/92 $5.00 Copyrlght C 1992 by Academic Press, Inc All rights of reproduction in any form reserved.
196
MCCAIN
AND
Wisconsin-Madison. The cottonwood that we sampled was one of the same trees that Russin and Evert (1984, 1985) had used previously for leaf structure studies. By using a sharp cork borer, 4-mm leaf disks were excised from near the centers of leaf blades as distant as possible from large veins. The disks were placed in a sample holder that was designed to ensure magnetic field homogeneity and to orient the leaf disk with its surface perpendicular to the static magnetic field and to the pulsed field gradient (McCain et al., 1984; McCain, 1986). ‘H image spectra were obtained at 400 MHz on a Bruker AM400 spectrometer equipped with a Bruker microimaging probe. Technical details concerning operation of the instrument have been published separately in a specialized magnetic resonance journal (McCain et al., 1991); briefly, the experiment required that field gradients and the NMR transmitter be turned on and off to produce a timed sequence of pulses. The NMR signal was Fourier transformed in two dimensions and tilted to properly align the chemical shift and image axes. We call the resulting 2-D contour plot an “image spectrum” because one axis contains pure image information, while the other axis is equivalent to the NMR spectrum. RESULTS
AND
DISCUSSION
Signal intensity in the high-resolution iH NMR spectrum of a leaf arises almost entirely from H,O; other hydrogen nuclei produce either very broad peaks (because they are in biopolymers or membranes) or much less intense peaks (because they are at lower concentrations than water). Therefore, an NMR image reveals primarily the distribution of water. However, a leaf spectrum is more complex than the single, narrow peak that is obtained from water because internal structures in the leaf distort the applied magnetic field; these effects in turn distort the image. Magnetic heterogeneity from internal air spaces broadens the signals and degrades the resolution. Indeed, image resolution is so poor in well-aerated leaves that there is little hope to resolve individual cells unless the cells are very large (Walter et al., 1989). Another magnetic effect causes NMR signals from chloroplast water to be displaced from those of water in the other leaf compartments (McCain and Markley, 1986). Chemical shifts can distort conventional NMR images (McCain and Markley, 1990), although the distortion seldom is serious in most biological samples because the shifts are small. However, leaves are exceptional; often in leaves the water signals exhibit a wide range of chemical shifts. By including chemical shift as a separate dimension, our technique eliminates shift distortion. An NMR image spectrum contains information about the distribution of water and chloroplasts in a leaf. Consider the examples shown in Fig. 1. Each of the six contour plots is distinctly different from the others, yet the patterns are essentially reproducible. We obtained similar results in repeated samplings from the same species. Since the image spectra are species specific, it is clear that the technique can detect internal structural differences. This paper
MARKLEY
ti
.,\‘-_i ,A \.-__ :/, I/
Populus
deltoides
----,’
image spectra from six species. An image specFIG. 1. NMR trum is a two-dimensional contour plot that shows how water is distributed in a leaf. The vertical axis is a chemical shift dimension, while the horizontal axis represents depth into the leaf along a line perpendicular to the surface. The upper surface lies to the left in all six image spectra. The L-shaped figure at lower left shows the scale for all plots; its horizontal bar corresponds to 50 km, and its vertical extension is equivalent to 2 ppm (parts per million) of chemical shift. This figure demonstrates that leaves with different internal structures produced quite different image spectra.
shows how to interpret the data to study water distribution. A future paper will consider chloroplast distribution. By integrating an image spectrum along the spectrum dimension, we obtain what we call a “water profile,” so named because it shows the onedimensional (1-D) cross-sectional distribution of all water in the leaf regardless of its chemical shift. Figure 2 shows the relationship between the image spectrum, the water profile, and the spectrum. The water profile and the spectrum are each 1-D representations of the image spectrum but with all information deleted from the other dimension. Although the spectrum can be obtained by integrating the image spectrum along the depth dimension, it can be recorded also in a simple NMR experiment with no field gradient applied. However, the undistorted water profile can be obtained only by integrating the 2-D image spectrum along the chemical shift dimension. A simple 1-D NMR imaging experiment would not remove the chemical shift distortion (McCain and Markley, 1990). Our experimental water profiles exhibit poor resolution. The problem is apparent in the smooth, rounded water profile shown in Fig. 2 and reproduced (as a solid-line curve) in Fig. 3. A perfectly resolved profile (to which the heavy dotted-line curve in Fig. 3 is a close approximation) would be
NMR
IMAGING
STUDY
197
OF LEAVES
I 50
0 Depth
.\_ I
I
(
0
I,
Depth
I
OF STRUCTURAL
A Nuclear
BIOLOGY
108,
1%%201
(19%)
Magnetic Resonance Imaging Technique Studies of Water in Plant Leaves DOUGLAS
*Department
lJniuersLt+y Uniuerssty
of Southern Mississippi, of Wisconsin, Madison,
Received April 29, 1991, and in revised form February
A new imaging technique is described which uses nuclear magnetic resonance (NMR) to create a water profile of plant leaves. The water profile shows the average distribution of water as a function of depth into a leaf along a line perpendicular to the leaf surface: it can be used to measure the thickness of cell layers and the quantity of water in each layer. Two-dimensional NMR methods were used to avoid chemical shift distortion which degrades the resolution in leaf images made by conventional NMR techniques; image resolution was improved further by deconvolution analysis. To illustrate its application, the technique was used to follow changes in the internal structure of developing leaves. Q 1992 Academic Press, Inc.
INTRODUCTION
Nuclear magnetic resonance (NMR) techniques can be used to create images that show the distribution of resonating atomic nuclei in a sample. When the sample is living tissue and the instrument is tuned to hydrogen nuclei, the images show predominantly the distribution of liquid water. Medical applications of NMR imaging are well developed (Moonen et al., 19901, and the technique is beginning to be used for botanical research (see, for example, Veres et al., 1991). Raw data from a typical NMR imaging experiment consist of three-dimensional information encoded in a computer memory; the usual practice is to view selected two-dimensional (2-D) slices through the data set. However, NMR imaging is not limited to the three Cartesian axes of geometrical space. Any number of dimensions can be imaged, and the dimensions may include nongeometrical NMR variables. We have developed a 2-D NMR imaging technique in which one dimension represents depth into a layered sample ialong an axis perpendicular to the ’ To whom correspondence
should be addressed.
for
AND JOHN L. MARKLEY?
C. McCAIN*,I
of Chemistry and BiochemLstry, and +Biochemistry Department,
Designed
Hattiesburg, Wisconsin
Mississippa 53706
39406;
1, 1992
layer surfaces), while the other dimension shows chemical shift (a magnetic effect related to the chemical environment of the nuclei) (MeCain et al., 1991); the data are presented as contour plots that show how water in the sample is distributed across the two dimensions. The technique was designed specifically for studies of plant leaves. By including a chemical shift dimension, the method cancels certain distortions that would blur a purely geometrical image, and it produces information that-can be used to distinguish chloroplast water from water that is not located in the chloroplasts. By restricting the number of geometrical dimensions to one, the method focuses on the average distribution of water among cell layers in a planar, layered leaf. We foresee at least five applications of the 2-D imaging technique to studies of plant leaves. The technique can provide (1) images that show the distribution of water as a function of depth into the leaf, (2) separate images of chloroplast water and nonchloroplast water, (3) separate images of vacuolar water and nonvacuolar water, (4) data related to the movement, diffusion or exchange of water between adjacent cells or subcellular compartments in the leaf, and (5) information about the distribution of bound water in a leaf. The objective of this paper is to introduce the new technique. By concentrating on the first application listed above, we describe how water profiles are obtained and discuss how they may be interpreted. We then illustrate the use of water profiles in a study of leaf development. Future papers will discuss the other applications. EXPERIMENTAL Healthy leaves were harvested from the following species: Norway maple (Acer plutunoides L. cv. “Emerald Queen”), lilac, (Syringa uulgaris L. cv. “Madame Lemoine”), cotton (Gossypium hirsutum L. cv. “Texas Marker 1’7, willow (Salti alba L. cv. “Vitellina”), tulip tree (Liriodendron tulipifera L.), and cottonwood Populus deltoides Bartr. ex Marsh). The cotton plant was grown outdoors in a pot; others grew on the campus of the University of
1047-8477/92 $5.00 Copyrlght C 1992 by Academic Press, Inc All rights of reproduction in any form reserved.
196
MCCAIN
AND
Wisconsin-Madison. The cottonwood that we sampled was one of the same trees that Russin and Evert (1984, 1985) had used previously for leaf structure studies. By using a sharp cork borer, 4-mm leaf disks were excised from near the centers of leaf blades as distant as possible from large veins. The disks were placed in a sample holder that was designed to ensure magnetic field homogeneity and to orient the leaf disk with its surface perpendicular to the static magnetic field and to the pulsed field gradient (McCain et al., 1984; McCain, 1986). ‘H image spectra were obtained at 400 MHz on a Bruker AM400 spectrometer equipped with a Bruker microimaging probe. Technical details concerning operation of the instrument have been published separately in a specialized magnetic resonance journal (McCain et al., 1991); briefly, the experiment required that field gradients and the NMR transmitter be turned on and off to produce a timed sequence of pulses. The NMR signal was Fourier transformed in two dimensions and tilted to properly align the chemical shift and image axes. We call the resulting 2-D contour plot an “image spectrum” because one axis contains pure image information, while the other axis is equivalent to the NMR spectrum. RESULTS
AND
DISCUSSION
Signal intensity in the high-resolution iH NMR spectrum of a leaf arises almost entirely from H,O; other hydrogen nuclei produce either very broad peaks (because they are in biopolymers or membranes) or much less intense peaks (because they are at lower concentrations than water). Therefore, an NMR image reveals primarily the distribution of water. However, a leaf spectrum is more complex than the single, narrow peak that is obtained from water because internal structures in the leaf distort the applied magnetic field; these effects in turn distort the image. Magnetic heterogeneity from internal air spaces broadens the signals and degrades the resolution. Indeed, image resolution is so poor in well-aerated leaves that there is little hope to resolve individual cells unless the cells are very large (Walter et al., 1989). Another magnetic effect causes NMR signals from chloroplast water to be displaced from those of water in the other leaf compartments (McCain and Markley, 1986). Chemical shifts can distort conventional NMR images (McCain and Markley, 1990), although the distortion seldom is serious in most biological samples because the shifts are small. However, leaves are exceptional; often in leaves the water signals exhibit a wide range of chemical shifts. By including chemical shift as a separate dimension, our technique eliminates shift distortion. An NMR image spectrum contains information about the distribution of water and chloroplasts in a leaf. Consider the examples shown in Fig. 1. Each of the six contour plots is distinctly different from the others, yet the patterns are essentially reproducible. We obtained similar results in repeated samplings from the same species. Since the image spectra are species specific, it is clear that the technique can detect internal structural differences. This paper
MARKLEY
ti
.,\‘-_i ,A \.-__ :/, I/
Populus
deltoides
----,’
image spectra from six species. An image specFIG. 1. NMR trum is a two-dimensional contour plot that shows how water is distributed in a leaf. The vertical axis is a chemical shift dimension, while the horizontal axis represents depth into the leaf along a line perpendicular to the surface. The upper surface lies to the left in all six image spectra. The L-shaped figure at lower left shows the scale for all plots; its horizontal bar corresponds to 50 km, and its vertical extension is equivalent to 2 ppm (parts per million) of chemical shift. This figure demonstrates that leaves with different internal structures produced quite different image spectra.
shows how to interpret the data to study water distribution. A future paper will consider chloroplast distribution. By integrating an image spectrum along the spectrum dimension, we obtain what we call a “water profile,” so named because it shows the onedimensional (1-D) cross-sectional distribution of all water in the leaf regardless of its chemical shift. Figure 2 shows the relationship between the image spectrum, the water profile, and the spectrum. The water profile and the spectrum are each 1-D representations of the image spectrum but with all information deleted from the other dimension. Although the spectrum can be obtained by integrating the image spectrum along the depth dimension, it can be recorded also in a simple NMR experiment with no field gradient applied. However, the undistorted water profile can be obtained only by integrating the 2-D image spectrum along the chemical shift dimension. A simple 1-D NMR imaging experiment would not remove the chemical shift distortion (McCain and Markley, 1990). Our experimental water profiles exhibit poor resolution. The problem is apparent in the smooth, rounded water profile shown in Fig. 2 and reproduced (as a solid-line curve) in Fig. 3. A perfectly resolved profile (to which the heavy dotted-line curve in Fig. 3 is a close approximation) would be
NMR
IMAGING
STUDY
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OF LEAVES
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