Journal of Experimental Botany, Vol. 51, No. 353, pp. 2109-2117,
December 2000
© 2000 Oxford University Press
Metabolite monitoring in plants with double-quantum filtered chemical shift imaging
1 Institut für Organische Chemie, Am Hubland, D-97074 Würzburg, Germany
2 Physikalisches Institut, Am Hubland, D-97074 Würzburg, Germany
3 Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
4 Institut für Pharmazie und Lebensmittelchemie, Am Hubland, D-97074 Würzburg, Germany
Received 24 March 2000; Accepted 11 July 2000
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Abstract |
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1H spectroscopic imaging in combination with double-quantum filtering with magnetic field gradients is used for in vivo applications in humans and animals. Because of its high selectivity and strong reduction of water signal it is also a useful tool for monitoring the distribution of specific metabolites in plants. The development and application of a double-quantum selective spectroscopic imaging sequence for detecting the sucrose distribution in the stem of sugar cane (Saccharum officinarum L.) is described. The results show that local differences in sucrose distribution can be detected non-invasively with a resolution of 0.4x0.4x6 mm3 to 0.645x0.645x6 mm3.
Key words: NMR imaging, localized NMR spectroscopy, metabolite monitoring, sugar cane.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In vivo NMR spectroscopic imaging methods allow non-invasive metabolic studies in living organisms. New developments in these techniques, mainly used in clinical and pharmacological research, have prepared the ground for successful applications on plants (Walter et al., 1992
; Ratcliffe, 1994; McFall and Van As, 1996; Chudek and Hunter, 1997). MR data available from living plants range from anatomical and physiological parameters, like water distribution (Kuchenbrod et al., 1995; Meininger et al., 1997b) and flow velocities (Van As and Schaafsma, 1984; Köckenberger et al., 1997; Edzes et al., 1998; Kuchenbrod et al., 1998; Scheenen et al., 1998; Rokitta et al., 1999), to the monitoring of metabolites (Ziegler et al., 1996; Meininger et al., 1997a; Verscht et al., 1998).
Metabolite detection using 1H NMR spectroscopy in plants is difficult to accomplish because of the dominating presence of water and a large overlap in metabolite signals. Water usually has a significantly broader linewidth in plant tissues as compared to animal tissues, because of the prominent presence of magnetic susceptibility gradients. The water signal needs to be suppressed to enable detection of other 1H-containing compounds, which generally have a concentration that is smaller than that of water, by a factor of 1000. Additionally, plant tissue contains a large variety of different 1H-containing metabolites (Fan et al., 1986), creating complex spectra, partly due to the large amount of signal overlap. If there is interest in only one specific assimilate, a method selectively to detect only that compound would be preferable.
Both water suppression and a partial selection of specific metabolites can be achieved by multiple-quantum (usually zero- and double-quantum) filtering using phase cycling (Hore et al., 1982) or pulse field gradients (Wokaun and Ernst, 1977; Bax et al., 1980; van Dijk et al., 1992) for coherence pathway selection. Multiple-quantum editing techniques have, for example, been used for the selective detection of lactate in tumours in the presence of lipid signals (Sotak et al., 1988; Trimble et al., 1990; Lei and Peeling, 1999). DQ filtered spectroscopic imaging has been demonstrated on tissue samples and rat brain (He et al., 1995, 1996).
The aim of this study was to realise a spectroscopic imaging experiment on an intact plant with water suppression and signal editing of specific metabolites using double-quantum filtering. Metabolite resonances near the water line can be better resolved using this method. A double-quantum filtered spectroscopic imaging sequence adapted for monitoring assimilates in plants is described and, as a first application, the spatial distribution of sucrose in the stem of a sugar cane plant was measured. Sucrose is a good subject for testing the sequence because it has resonances very close to that of water, making the signal sensitive to the quality of the water suppression. Additionally, it is a vital assimilation product for the plant and usually present in high quantities (Burr et al., 1957; Batta and Singh, 1986).
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Materials and methods |
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Plant material
Sugar cane (Saccharum officinarum L.) was grown from pith cuttings in a sand culture watered with standard nutrient solution (Hoagland and Arnon, 1938
) under greenhouse conditions. The average temperature was kept at 24 °C (minimum 20 °C, maximum 27 °C), and the average humidity at 69% (minimum 50%, maximum 82%). Light intensity was kept at an average of 420 µmol m-2 s-1 PAR between 6 h and 18 h. Plants used for NMR imaging were 6–12-weeks-old, reaching pith diameters from 10–20 mm. Light microscopy and autofluorescence images were performed with hand-cut cross-sections of the stem from the same plant under epi-illumination with a Leica Binocular MZFL III (Leica Microsystems, 33578 Wetzlar, Germany). Magnification was 6x and UV excitation followed by 395 nm.
NMR hardware
The NMR spectroscopy and imaging experiments were performed using a Bruker BIOSPEC 70/20 NMR spectrometer with a magnetic field strength of 7 T and a horizontal bore of 20 cm (Bruker Medical GmbH, Silberstreifen, 76287 Rheinstetten, Germany). The actively shielded gradient coils were capable of achieving gradients up to 0.2 T m-1 with rise and fall times of 240 µs. For excitation and detection of the NMR signal at the 1H resonance frequency of 300.3 MHz, two different home-built coils were used. One, a Helmholtz coil, has two rings with a diameter of 20 mm spaced 10 mm apart, and was used for experiments on phantoms and thin sugar cane stems. The other, the birdcage resonator, is a high pass design with four rungs, a diameter of 24 mm, and a length of 30 mm. Measurements on thick sugar cane stems were made with this coil.
DQ spectroscopic imaging
A double-quantum (DQ) filter was constructed with imaging capabilities to obtain a DQ filtered spectroscopic imaging sequence (Fig. 1).
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The pulse programme starts with a 90°–180°–90° pulse series to generate double-quantum magnetization that shows no chemical shift dependent modulation, due to refocussing by the 180° pulse. Spoiler gradients were applied before and after the 180° pulse to remove all unwanted signals caused by imperfect pulse angles. The optimal echo time between the 90° pulses to obtain maximal double-quantum coherence of spins coupled with a coupling constant J is 1/2J. During the delay between the second and the third 90° pulse, the evolved coherences are dephased by switching on editing gradients, which will be rephased later in the sequence. The 180° pulse during this period reduces the effects of double-quantum frequency modulation on the final signal.
Conversion of the double-quantum coherences into observable magnetization is achieved by the third 90° pulse. This pulse consists of a 45°–
–45° pulse, a so-called 11-pulse (Levitt, 1986) which can in principle be made selective for certain frequencies by varying the period . The last 180° pulse removes frequency modulation of the signal before acquisition, serves as a slice selection pulse, and provides extra time for switching of the imaging and DQ filtering gradients. As the sensitivity of a coherence for gradient fields is proportional to its order, selective filtering of double-quantum coherences is achieved by dephasing and rephasing gradients with areas in the ratio 1:(–2). Without the slice-selective 180° pulse and the imaging gradients the sequence acts as a purely spectroscopical filter. Adding gradients for phase encoding in the two residual dimensions extends the sequence to a double-quantum filtered chemical shift imaging (DQ-CSI) experiment. An odd number of phase encoding steps (31x31) was chosen to allow symmetrical switching of the phase encoding gradient around the zero point, resulting in 961 single acquisitions for one complete DQ-CSI data set without averaging (FOV 8x8 or 20x20 mm, slice thickness 6 mm). An eight-step phase cycle was applied to diminish phase artefacts. The DQ echo time (which here is the sum of the delay between the first and the second 90° pulse and the delay between the third 90° pulse and the start of acquisition) was set to 70 ms unless stated otherwise. The period between the second and the third 90° pulse lasted 11.5 ms. The repetition delay was 0.5 s for the DQ-CSI experiment, resulting in a total experimental time of 101 min for eight averages.
A standard two-dimensional cross-sectional spin-echo imaging sequence with a matrix size of 128x128 picture elements was used for preparation of the experiments. The 90° and 180° pulses were slice-selective 500 µs Gauss-shaped pulses. Spoiler gradients were applied before and after the 180° pulse. The slice thickness was 1 mm, the FOV was, as in the DQ-CSI images, 8 mm for the phantom image, and 20 mm for the sugar cane image. The echo time was 7.8 ms, and the repetition delay was set to 1.0 s.
All spectra were recorded with a sweepwidth of 5 kHz and 8 k data points.
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Results |
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Phantom experiments
The double-quantum filter (Fig. 1
) was tested on an aqueous solution of saccharose (0.5 M) (Fig. 2) without spatial encoding.
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Figure 3
shows the global proton spectrum without (Fig. 3A) and with (Fig. 3B) filtering.
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The signal of the saccharose protons at C-1', which do not own a direct coupling partner (Fig. 2
) and are thus not subject to double-quantum transitions, have disappeared from Fig. 3B.
To optimize the double-quantum echo time, spectra with echo times from 40–90 ms were recorded (Fig. 4).
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Saccharose resonances were obtained between 40 and 80 ms, corresponding to coupling constants between 6.3 and 12.5 Hz. The echo time of 70 ms (J=7.1 Hz) was chosen for all further experiments as it seemed to offer the best compromise between signal quality and baseline distortion.
Next, spatial encoding gradients were added to the pulse programme, and the double-quantum chemical shift imaging (DQ-CSI) sequence was applied to a radial phantom with the outer tube filled with 0.5 M of an aqueous solution of saccharose and the inner one filled with water. The proton spin-echo image (Fig. 5A) shows the cross-sectional water distribution of this phantom. In Fig. 5B, results of the DQ-CSI data set are shown. The image displays the spatial distribution of the double-quantum filtered saccharose signal, which is obtained by integration of the corresponding spectral region from 3.0–4.5 ppm. The spectrum shown (in magnitude mode) belongs to the volume element marked with a circle in the image. In the centre, where no saccharose is present, the signal intensity between 3.0–4.5 ppm does not rise above the noise level.
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Sugar cane (Saccharum officinarum)
Global in vivo spectra (Fig. 6) were recorded from the first internodium of the sugar cane S.officinarum without filtering (Fig. 6A, B), with CHESS water suppression (Fig. 6C, D), and with double-quantum filtering (Fig. 6E, F).
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The number of averages was set to eight for the spectra on the left (Fig. 6A
, C, E), and was increased to 128 for the spectra on the right (Fig. 6B, D, F). As expected, the global, unfiltered proton spectra Fig. 6A and B contain a dominating water signal. Suppressing this with a CHESS sequence (Fig. 6C, 6D) leads to a better visibility of the sugar resonances in the range of 3–4 ppm, and the ratio between the peak heights of the highest saccharose signal (in absolute mode) and that of water was improved from about 4%/0.04 in Fig. 6B to 11%/0.11 in Fig. 6D. The double-quantum filtered spectrum Fig. 6E, although having a low signal-to-noise ratio (SNR) of about 13 (calculated from the maximum signal height versus the standard deviation of the noise), shows almost complete water suppression, and the in vivo double-quantum part of the sugar resonances is clearly visible as a broad signal between 3 and 4 ppm. Increasing the number of averages to 128 led to an improvement of the SNR to about 40. The ratio between the peak heights of the highest saccharose signal and that of water was improved again and reached 160%/1.6 in Fig. 6F.
Figure 7 shows light microscopy sections from the first internodium of the sugar cane stem.
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Figure 7A
was made under epi-illumination, Fig. 7B shows UV autofluorescence whilst Fig. 7C was made under epi-illumination after staining with phloroglucinol/ HCl. In all images, the central cavity can be distinguished. Furthermore, Fig. 7A shows bright autofluorescence of highly lignified vascular bundles which appear red after staining in Fig. 7C. The average diameter of the vascular bundles was found to be 30 µm.
The application of the DQ-CSI sequence on an intact sugar cane stem is shown in Fig. 8.
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For comparison, a cross-sectional spin-echo image (Fig. 8A
) was recorded from the first internode, showing the distribution of the proton signal with a resolution of 156x156 µm2. The DQ-CSI experiment provided the distribution of the metabolite double-quantum signal (Fig. 8B) obtained by integration between 3 and 4.5 ppm, i.e. in the typical region of sugar resonances (see Fig. 4). The corresponding double-quantum filtered spectra are shown for selected voxels 1–3 in phase-sensitive mode. No reasonable NMR resonance was detected in the middle of the stem, and the spin-echo image shows this central region to give a poor water signal as well. The brightest structures, however, found in the spin-echo image 8A correspond well to the highly lignified vascular bundles identified in the stained light microscopy image (Fig. 7C), although they were made from slightly different slices. The DQ-CSI experiment (Fig. 8B), with an in-plane resolution of 645x645 µm2, cannot resolve such fine structures as the vascular bundles, but is capable of providing a more global metabolite map instead. Metabolites showing double-quantum resonances between 3 and 4.5 ppm appear to be concentrated in the region between the pith and the cortex of the sugar cane stem. With 31x31 phase-encoding steps and 32 averages, the total experimental time was 404 min. The NMR resonances of sugar-type metabolites and water are clearly separated in the spectra. Increasing the number of averages improves the SNR in the spectra (Fig. 9), but the elongation of the total experimental time needs to be kept in mind. The DQ spectrum of a selected voxel has an SNR of 11 for eight averages (Fig. 9A), of 22 for 32 averages (Fig. 9B), and of 25 for 64 averages (Fig. 9C), recorded in 1.5, 6, and 12 h, respectively.
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Discussion |
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Metabolite monitoring is a challenging task for in vivo proton NMR spectroscopy and imaging in plants. A double-quantum filter enables both suppression of the water signal and selective detection of certain metabolites. The pulse programme described here is capable of specifically selecting NMR signals that arise from double-quantum transitions. This was demonstrated on a saccharose phantom by the disappearance of the single-quantum signals of the saccharose 1' protons when applying the filter (Fig. 3
). A further benefit offered by the method is the possibility of selecting metabolites of interest by tuning the double-quantum echo time for the selection of distinct coupling constants of interest. Monitoring the spatial distribution of selected signals was achieved by extending the sequence by phase-encoding gradients to a working DQ-CSI experiment. This way a distribution of the monitored saccharose signal in the object can be compared with the corresponding spin-echo image (Fig. 5). The first application of double-quantum filtered spectroscopy on a living plant was done by detecting the double-quantum signal of metabolites in the spectral region between 3 and 4.5 ppm with a maximum sensitivity for protons with couplings of 7.1 Hz in sugar cane (Fig. 6) and monitoring their spatial distribution in the stem with the DQ-CSI sequence with a resolution of 0.645x0.645x6 mm acquired in 6.7 h (Fig. 8). An assignment of the observed peaks to different contributing compounds based on the one-dimensional spectra only is difficult due to the large in vivo line width. According to the literature, the main metabolite in sugar cane showing the selected resonances is saccharose, followed by glucose and fructose, which are present in the stem in quantities of about 70, 20, and 20 mg g-1 fr. wt, corresponding to 0.204, 0.11 and 0.11 mmol g-1 fr. wt, respectively, in this state of growth (Batta and Singh, 1986). Thus, the monitored signal most likely represents the distribution of the double-quantum resonances of these free sugars—especially saccharose, claiming half of the molarity of the free sugar content—in the sugar cane plant. As double-quantum coherences are more prone to relaxation processes than single-quantum coherences, local differences in the magnetic susceptibility (e.g. between two materials such as air space and cell walls) may cause more severe differences in double-quantum relaxation rates. This may generate additional relaxation contrast in the images, which may not represent a pure spin-density map (and thus the actual distribution of the metabolites) any more. Besides partial volume effects, relaxation phenomena may be responsible for the decreasing DQ signal towards the inner and outer boundaries in Fig. 8B.
For monitoring metabolites in plant seedlings, a spatially resolved COSY (correlated spectroscopy) experiment (correlation peak imaging CPI) was developed, providing the localization of preselected COSY cross-peaks for slices of 4 mm by a matrix of 16x16 pixel at a field of 500 MHz with a sampling time of 4 h 33 min without averaging (Ziegler et al., 1996). This experiment is suitable for static systems, as even slow changes in the sample will lead to a loss of information. The study of slow changes in plants such as the kinetics of sucrose uptake in a seedling was measured by a CSI experiment at 500 MHz (Verscht et al., 1998). The nominal resolution of 0.125x0.125x2 mm3 was achieved within 70 min. For bigger plants than seedlings, a wider bore and thus often a lower magnetic field and other detection coils are required, causing the resolution to become worse.
A CSI sequence with CHESS (chemical shift selective) water suppression for metabolite detection was used on 2-year-old lianas at 300 MHz and achieved spatially resolved spectra with a resolution of 0.21x0.21x2 mm3 for a 64x64 pixel matrix in 11 h, one shot lasting 86 min (Meininger et al., 1997b). Compared with the resolution and spectral quality of Meininger, the acquisition time for the DQ-filtered CSI still appears to be quite long due to the intrinsic loss of signal by choosing DQ coherences only and omitting all other coherence pathways, and due to relaxation processes. The advantage of DQ-CSI lies in the possibility of selecting couplings of interest in one spectral dimension by simply varying a delay, thus being a method between the highly selective, but time-consuming CPI and the non-selective standard CSI sequence.
Furthermore, sucrose, although the main assimilate in sugar cane, was not an optimal substrate for the detection of DQ coherences. The spin systems in sucrose are far from simple compared to, for example, lactate, which was enhanced and monitored selectively (He et al., 1995, 1996). None of the saccharose protons are magnetically equivalent. The saccharose resonances lie close to each other and are reduced in signal height due to splitting and coupling to more than one partner. The resonances also show overlap under in vivo conditions because of broad line widths. Large signal losses occur due to overlap between resonances that have opposite phases, caused by differences in their DQ modulations. Thus, a better yield of DQ signal can be achieved by focusing on other assimilates or metabolites with well-defined AX spin systems and stronger couplings. While such AX systems enable selective enhancement by using a selective 180° pulse between the second and the third 90° pulse, strong coupling allows the use of shorter DQ echo times (proportional to 1/2J) causing less signal losses due to relaxation.
Finally, the spatial monitoring of the DQ signal can be improved by using different sampling methods, for example, by acquisition-weighted CSI (Mareci and Brooker, 1984, 1991) as realized in SLOOP (spectral localization with optimal pointspread function) (Kienlin and Mejia, 1991) or by radial sampling (Meininger et al., 1997a), thus reducing acquisition time.
Already, the DQ-CSI experiment offers a non-invasive method for metabolite monitoring on living plants, capable of repeated and long-time measurements of the distribution of assimilates in selected plant areas. A good example of this is demonstrated in Fig. 8, where the DQ signal of mainly saccharose protons is very low in the middle of the pith and the cortex and highest in between those two regions, yielding a ring-shaped pattern. Changes in assimilate distribution may thus be measured in different environmental conditions, during growth and development. DQ-CSI as an in vivo NMR experiment provides plant physiologists with a new tool to study adaptation processes.
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Acknowledgments |
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This work was funded by the Deutsche Forschungsgemeinschaft (‘Graduiertenkolleg NMR in vivo und in vitro für die biologische und medizinische Grundlagenforschung’) and the Fonds der Chemischen Industrie. KW thanks the Studienstiftung des Deutschen Volkes and AvdT the European Union for generous fellowships. The authors wish to thank Markus Rokitta, Michael Szimtenings, Wilfried Landschütz, and Titus Lanz for designing and building the probeheads and Dr Markus Rückert for fruitful discussions.
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Notes |
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5 To whom correspondence should be addressed. Fax: +49 931 888 4755. E-mail: bringman{at}chemie.uni\|[hyphen]\|wuerzburg.de
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Abbreviations |
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CHESS, chemical shift selective; COSY, correlated spectroscopy; CPI, correlation peak imaging; CSI, chemical shift imaging; DQ, double-quantum; NMR, nuclear magnetic resonance; SLOOP, spectral localization with optimal pointspread function.
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References |
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