Computed

Dec 23, 2010 - Vanessa Gómez-Vallejo , Vijay Gaja , Kiran B. Gona , Jordi Llop. Journal of Labelled Compounds and Radiopharmaceuticals 2014 57 ...
3 downloads 0 Views 5MB Size
Download PDF
Anal. Chem. 2011, 83, 578–584

Three-Dimensional Positron Emission Tomography/ Computed Tomography Analysis of 13NO3- Uptake and 13N Distribution in Growing Kohlrabi Wansheng Liang,†,‡ Yingchun Nie,† Jun Wang,‡ Jing Wu,‡ Hui Liu,‡ Qi Wang,‡ Lijuan Huang,† Hao Guo,§ Boxue Shu,§ and Jiagen Lv*,† Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Shaanxi Normal University, Xi’an, 710062, China, Changan Hospital, No. 17, Wenjing Road, Xi’an, 710016, China, and Xi’an Gaoxin Hospital, No. 16, South Tuanjie Road, Xi’an, 710075, China We report the application of three-dimensional positron emission tomography/computed tomography (PET/CT) for the analysis of 13NO3- uptake and 13N distribution in growing kohlrabi. The analytical procedures, equipment parameters, and image reconstruction mode for plant imaging were tested and selected. 13N in growing kohlrabi plants was imaged versus time using both PET movies and PET/CT tomograms. The 13NO3transport velocity in kohlrabi from root to petiole was estimated to be 1.0 cm/min. The appearance of shellshaped 13NO3- transport pathways, corresponding to the kohlrabi corm, suggests the existence of special routes with higher efficiency for 13NO3- transport, which tends to have the shortest distances to the leaves or buds. Standardized uptake values (SUVs), used as the representative figures for describing 13N distribution, were quantified versus time at some putative sites of interest. For multiple analysis of the same-plant, 13N distribution in kohlrabi under normal conditions, methionine sulfoximine (MSX) stress, and recovery from MSX stress was examined. The 13N distribution variation studies were also done under the above three growth conditions. Our results suggest a significant downregulation of nitrate uptake in kohlrabi in the presence of MSX. Positron emission tomography (PET) is one of the most sensitive molecular imaging modalities capable of generating realtime and whole-body/tissue three-dimensional (3D) imaging of living systems with temporal and spatial resolutions. In addition, positron emitting radioactive isotope-labeled probes can be chemically and biochemically indistinguishable from their nonradioactive counterparts,1 rendering any possible complications to experimental observations and conclusions introduced by molecule labeling negligible. Taking the molecular imaging concept of PET a step further is the combination of positron emission tomogra* To whom correspondence should be addressed. Phone: +86-29-85308442. Fax: +86-29-85307774. E-mail: [email protected]. † Shaanxi Normal University. ‡ Changan Hospital. § Xi’an Gaoxin Hospital. (1) Ametamey, S. M.; Honer, M.; Schubiger, P. A. Chem. Rev. 2008, 108, 1501– 1516.

578

Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

phy/computed tomography (PET/CT), which results in an optimal correlation of anatomic and metabolic information. Albeit young, the technique has already become a leading analytical tool in many research fronts, including the study of human cognition and central nervous system investigation,2-4 drug development,5 and cancer research.6 PET/CT imaging has mainly been applied to clinical tumor diagnosis and therapy.7,8 It has also generated increasing interest among chemists.1 Efforts to develop new positron probes,9 new synthesis methods for the development of positron emitting radioactive isotope-labeled probes, and positron imaging applications on microstructure investigations have been reported recently.10-14 Analyzing molecular uptake, transport, and distribution in growing plants is an important research area in the plant sciences. Plants are known to exhibit sensitive and systematic physiological responses to mechanical damage, biologic or abiotic invasions, and environmental changes. Thus, invasive analytical methods may result in ambiguous and even contradictory interpretations of experimental data. For example, the regulation of nitrate and ammonium uptake following the application of methionine sul(2) McNab, F.; Varrone, A.; Farde, L.; Jucaite, A.; Bystritsky, P.; Forssberg, H.; Klingberg, T. Science 2009, 323, 800–802. (3) Gordon, G. R. J.; Choi, H. B.; Rungta, R. L.; Ellis-Davies, G. C. R.; MacVicar, B. A. Nature 2008, 456, 745–749. (4) Thor, S. Nature 2008, 456, 177–178. (5) Eckelman, W. C.; Reba, R. C.; Kelloff, G. J. Drug Discovery Today 2008, 13, 748–759. (6) Niu, G.; Sun, X.; Cao, Q.; Courter, D.; Koong, A.; Le, Q.-T.; Gambhir, S. S.; Chen, X. Clin. Cancer Res. 2010, 16, 2095–2105. (7) Townsend, D. W. In Positron Emission Tomography Clinical Practice; Valk, P. E.; Delbeke, D.; Bailey, D. L.; Townsend, D. W.; Maisey, M. N., Eds.; Springer: London, 2006; pp 1-16. (8) Costelloe, C. M.; Rohren, E. M.; Madewell, J. E.; Tsuyoshi, H.; Theriault, R. L.; Yu, T.-K.; Lewis, V. O.; Ma, J.; Stafford, R. J.; Tari, A. M.; Hortobagyi, G. N.; Ueno, N. T. Lancet Oncol. 2009, 10, 606–614. (9) Matson, J. B.; Grubbs, R. H. J. Am. Chem. Soc. 2008, 130, 6731–6733. (10) Ting, R.; Harwig, C.; Keller, U.; McCormick, S.; Austin, P.; Overall, C. M.; Adam, M. J.; Ruth, T. J.; Perrin, D. J. Am. Chem. Soc. 2008, 130, 12045– 12055. (11) Shokeen, M.; Anderson, C. J. Acc. Chem. Res. 2009, 42, 32–841. (12) Lee, C.-C.; Sui, G.; Elizarov, A.; Shu, C. J.; Shin, Y.-S.; Dooley, A. N.; Huang, J.; Daridon, A.; Wyatt, P.; Stout, D.; Kolb, H. C.; Witte, O. N.; Satyamurthy, N.; Heath, J. R.; Phelps, M. E.; Quake, S. R.; Tseng, H.-R. Science 2005, 310, 1793–1796. (13) Wallenborg, S.; Velikyan, I.; Bergstrolm, S.; Djodjic, M.; Ljung, J.; Berglund, O.; Edenwall, N.; Markides, K. E.; Langstrolm, B. Anal. Chem. 2004, 76, 7102–7108. (14) Bergstrolm, S. K.; Edenwall, N.; Laven, M.; Velikyan, I.; Langstrolm, B.; Markides, K. E. Anal. Chem. 2005, 77, 938–942. 10.1021/ac102510f  2011 American Chemical Society Published on Web 12/23/2010

foximine (MSX) to plant roots has been debated. Downregulation of nitrate and ammonium uptake has been suggested by some studies, but the opposite conclusion has also been reached by others.15,16 In most cases of investigations, regarding the biological processes in growing plants, such as the response to environmental changes or the metabolic variation of a molecule of interest, continuous or multiple analysis of the same-plant individual is preferable. Molecular imaging modalities have been extensively used for the visualization, characterization, and measurement of biological processes at the molecular and subcellular levels.1 Considering the aforementioned unique attributes of PET/CT and its demonstrated success in animal and human investigation, PET/ CT imaging could be a solution to the problems encountered in the analysis of growing plants. Positron-emitting radioactive isotopes have been exploited to study the metabolism of molecules of interest in higher plants by counting the radioactivity with a liquid scintillation counter,17 or by planar imaging of labeled molecules with a scintillation camera.18 Significant understanding of plant nutrition has also been achieved through these efforts. However, 3D PET/CT imaging of biomolecules or nutrients in plants has not yet been reported, and the envisioned advantages of PET/CT imaging for the biological analysis of plants have not been realized. The primary goal of this study is to extend the application of PET/CT imaging into analyzing nutrient molecules in growing higher plants to develop new applications for this technique. Under normal, aerated soil conditions, nitrate is the predominant form of nitrogen absorbed by plants regardless of the applied nitrogen source.19,20 Extensive research regarding nitrate metabolism has been done to study the effects of stress factors on plant growth.21 In this work, the 13NO3- uptake and 13N distribution in growing kohlrabi were analyzed, on the one hand, for the importance of nitrate for plant growth and, on the other hand, for some comparison of the results obtained from PET/CT with those from the literature. Kohlrabi plants were solution-cultured for the PET/CT analysis. Imaging analysis procedures, including the equipment parameters and accessory devices, were tested and constructed. Mimicking the same temperature and lighting conditions during the kohlrabi solution culture, we examined 13N in the growing kohlrabi through images reconstructed in forms of PET movies and PET/CT tomograms (in coronal, sagittal, and transaxial directions). This is the first time that the 13NO3- uptake and 13N distribution in growing plants have been visualized in 3D (15) Glass, A. D. M.; Britto, D. T.; Kaiser, B. N.; Kinghorn, J. R.; Kronzucker, H. J.; Kumar, A.; Okamoto, M.; Suman, R.; Siddiqi, M. Y.; Unkles, S. E.; Vidmar, J. J. J. Exp. Bot. 2002, 53, 855–864. (16) Britto, D. T.; Kronzucker, H. J. In Enhancing the Efficiency of Nitrogen Utilization in Plants; Goyal, S. S.; Tischner, R., Eds.; Haworth: New York, 2005; pp 1-23. (17) Kronzucker, H. J.; Siddiqi, M. Y.; Glass, A. D. M.; Kirk, G. J. D. Plant Physiol. 1999, 119, 1041–1045. (18) Kiyomiya, S.; Nakanishi, H.; Uchida, H.; Tsuji, A.; Nishiyama, S.; Futatsubashi, M.; Tsukada, H.; Ishioka, N. S.; Watanabe, S.; Ito, T.; Mizuniwa, C.; Osa, A.; Matsuhashi, S.; Hashimoto, S.; Sekine, T.; Mori, S. Plant Physiol. 2001, 125, 1743–1754. (19) Barker, A. V.; Bryson, G. M. In Handbook of Plant Nutrition; Barker, A. V.; Pilbeam, D. J., Eds.; CRC Press: Boca Raton, FL, 2007; pp 21-50. (20) Below, F. E. In Handbook of Plant and Crop Physiology, 2nd ed.; Pessarakli, M., Ed.; Marcel Dekker: New York, 2002; pp 385-406. (21) Girin, T.; El-Kafafi, E.-S.; Widiez, T.; Erban, A.; Hubberten, H.-M.; Kopka, J.; Hoefgen, R.; Gojon, A.; Lepetit, M. Plant Physiol. 2010, 153, 1250–1260.

modality. The 13NO3- transport velocity from the root to the petiole of kohlrabi was also estimated. The temporal 13N distributions among some putative sites (regions/tissues) of interest were studied by quantifying the relative radioactivity as the standardized uptake value (SUV) of these sites. For the multiple analyses of the same plant under different growth conditions, MSX was used to generate the stressed growth condition because of its potent inhibition of glutamine synthetase (GS). The 13NO3- uptake and 13N distribution under normal growth, MSX stress, and recovery from MSX stress conditions were imaged. Our results suggest downregulation of kohlrabi nitrate uptake was induced by MSX stress. EXPERIMENTAL SECTION Plant Culture. Vegetable crop kohlrabi (Brassica oleraces L. var. gongylodes L.) seeds were germinated in soil at room temperature. Following germination, plantlets were transferred to the field and cultured normally. After the size (diameter) of the plant’s corm reached about 2 cm, selected plants were transferred indoors and their roots were gently washed with tap water. Each plant was then placed in a 1 L light-shielded cylindrical polyethylene pot, containing 500 mL of Hoagland nutrient solution composed of the following: 4.0 mM Ca(NO3)2, 2.0 mM MgSO4, 4.0 mM KNO3, 0.4 mM (NH4)2SO4, 2 µM MnSO4, 0.3 µM CuSO4, 0.8 µM ZnSO4, 30 µM NaCl, 0.1 µM Na2MoO4, 1.43 mM KH2PO4, 10 µM H3BO3, and 20 µM Fe-Na-EDTA. Finally, the plants were cultured under artificial light at 16-18 °C. The culturing solution was replaced with fresh nutrient solution once a week. The aeration of the nutrient solution was conducted by pumping air into the solution continuously for 1 h at 1 h intervals. 13 NO3- Preparation. Following the previously reported method for 13NO3- synthesis,22,23 a 2 mL aqueous 13NO3- sample was produced in the cyclotron (Ion Beam Application, LouvainLa-Neuve, Belgium) by bombarding 16O water containing 0.5% 16 O hydrogen peroxide with 10-15 µA of 18 MeV protons. As previously reported,22-26 the cosynthesized contaminants included 13N2, 13NH4+, 13NO2-, trace 18F-, and 48V from the titanium beam entry window.22 Then, 13NO3- purification was conducted by following the procedures reported for plant 13 NO3- application.22,23 13NH4+ and 48V were eliminated by forcing the 13NO3- sample through an Alltech Maxi-Clean SCX cartridge (600 mg, Grace Davison Discovery Science, Deerfield, IL). Traces of 18F- was removed by passing the 13NO3- sample through a Sep-Pak Alumina N cartridge (500 mg, Waters Corporation, Milford, MA). The remaining 13NO2-, 13N2, and 16 O hydrogen peroxide were expelled by boiling the 13NO3sample, previously acidified with 0.5 mL of 0.5 mol/L H2SO4, for 1.5 min. After neutralizing with 0.75 mL of 0.5 mol/L cold K2CO3, the irradiation dose was measured using a CRC-15PET calibrator (J. Gravengaard Corporation, Portland, OR), and the (22) Wieneke, J.; Nebeling, B. Z. Pflanzenernaehr. Bodenkd. (J. Plant Nutr. Soil Sci.) 1990, 153, 117–123. (23) Siddiqi, M. Y.; Glass, A. D. M.; Ruth, T. J.; Fernando, M. Plant Physiol. 1989, 90, 806–813. (24) Tiedje, J. M.; Firestone, R. B.; Firestone, M. K.; Betlach, M. R.; Smith, M. S.; Caskey, W. H. Soil Sci. Soc. Am. J. 1979, 43, 709–716. (25) Tilbury, R. S.; Dahl, J. R. Radiat. Res. 1979, 79, 22–33. (26) Mcelfresh, M. W.; Meeks, J. C.; Parks, N. J. J. Radioanal. Chem. 1979, 53, 337–344.

Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

579

Figure 1. Photograph of a test plant fixed on the scanner bed and ready for PET/CT imaging. The upper right inset shows the content inside the lead pot. The root portion of the test kohlrabi was immersed in nutrient solution contained in a 60 mL end-sealed syringe, and 13 NO3- was injected into the syringe bottom via a polyethylene tubule. The lower left inset shows the locations of the seven putative sites of interest, where SUVs were measured under normal growth conditions.

measuring moment was recorded as t1. The final 13NO3solution was diluted with nutrient solution to 10 mL in a 20 mL syringe and was immediately delivered to the PET/CT scan room via a lead-shielded elevator. By controlling the proton bombarding time and beam current to about 15 min and 10-15 µA, respectively, the radioactivity of the final 13NO3- solution was controlled to a range of 2960 to 4440 MBq (80 to 120 mCi). Imaging Operation. A bracket was constructed to fix the kohlrabi plant onto the PET/CT scanner bed, as shown in Figure 1. It consisted of a polystyrene foam frame and a cylindrical hollow lead pot. The lead pot was 16 cm long and 11 cm in diameter with an end-sealed cavity 13 cm in length and 4 cm in diameter. The bracket and lead pot were fixed on the scanner bed with adhesive tape. A 60 mL syringe-made polypropylene tube (i.d. 3.0 cm), which fits the cavity size, was used to contain the plant root and nutrient solution during experiments. A 25 cm long and 1.5 mm i.d. polyethylene tubule with a Lure connector at the inlet end was used to deliver the 13NO3- solution to the bottom of the polypropylene tube. Prior to each experiment, the culturing solution was replaced with fresh nutrient solution, and the candidate plant was cultured at 16 °C for 3 h with the last 1 h being air charged continuously. The root portion was carefully inserted into the syringe-made polypropylene tube containing 10 mL of nutrient solution. The polypropylene tube was inserted into the lead pot cavity, and the plant was held by the polystyrene foam frame and adhesive tape. In the MSX stress experiment, solid MSX was weighed and dissolved in 500 mL of nutrient solution to prepare 1.0 mM MSX solution. Following the aforementioned operations, imaging was done by replacing the normal nutrient solution with the MSX solution. In all experiments, the tested plant was illuminated with 500 µmol m-2 s-1 artificial light. A scout CT scan (Discovery LS-PET/CT, GE Healthcare) was performed as an anatomic reference for the CT and PET scan that followed. This scout CT scan was also used to define the starting and ending locations for the PET/CT acquisitions. As the 580

Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

plant was fixed and scout CT scan was being done, the 13NO3was produced and purified. After 10 mL of 13NO3- solution had been injected into the syringe-made polypropylene tube, an additional 10 mL of the nutrient solution was immediately injected to wash out the residual 13NO3- in the polyethylene tubule. Then, the PET/CT scans were performed and the PET scan time was recorded as t2 for decay correction. PET images were acquired in 2D mode and reconstructed with 2D algorithm. The CT, PET, fused PET/CT images (in coronal, sagittal, and transaxial directions) and PET movies were generated using both the equipped Xeleris and the extra Mimvista (MIM Software Suite, MiMvista Crop.) workstations. Due to an unknown compatibility problem between Xeleris and Mimvista, a few of the reconstructed images could not be exported from Mimvista. For consistency, the PET/CT tomograms and PET movies, excluding specified ones, were exported from Xeleris, and the Mimvista reconstruction was used to aid image interpretation. SUV Measurement. SUV was used to normalize the supplied radiation dose, the decay correction, and the object weight difference for PET/CT analysis.27 It is currently a popular quantitative, easy-to-calculate, and noninvasive index of the metabolic rate of a labeled molecule.28,29 The SUV was calculated as follows:

SUV )

voxel value × weight decay-corrected dose

where the voxel value is scaled so that it represents MBq/cm3, the weight is the object weight in grams, and the decaycorrected dose is the supplied radiation dose corrected for decay from the positron radioactivity measuring time t1 to the PET scan time t2. The average SUV at a putative interest site (Figure 1), defined in a sphere of 3 mm diameter, was measured to represent the relative 13N amount. In normal growing plants, part of the absorbed nitrate can be rapidly converted into multiproducts, including nitrite, ammonium, amino acids, and even proteins; thus, the measured SUV should represent the total amount of 13 NO3- and its products. RESULTS AND DISCUSSION Analysis Procedures and Parameters. In the initial experiments, the test plant was fixed by an erectly standing polymethyl acrylate bed three days prior to the imaging. By moving the polymethyl acrylate bed, the test plant root was immersed in the 13 NO3- nutrient solution to complete the 13NO3- absorption. Root surface-bound 13NO3- nutrient solution was removed by washing with running tap water before the test plant and the polymethyl acrylate bed were placed on the scanner bed for PET/CT acquisition. A number of problems associated with such a procedure were quickly realized, including limited imaging frequency, inability to obtain real-time and continuous analysis, easily damaged plant samples, and most importantly, (27) Jadvar, H.; Parker, J. A. Clinical PET and PET/CT; Springer: London, 2005. (28) Lin, C.; Itti, E.; Haioun, C.; Petegnief, Y.; Luciani, A.; Dupuis, J.; Paone, G.; Talbot, J.-N.; Rahmouni, A.; Meignan, M. J. Nucl. Med. 2007, 48, 1626– 1632. (29) Thie, J. A. J. Nucl. Med. 2004, 45, 1431–1434.

Figure 2. Kohlrabi 13NO3- uptake and 13N distribution imaging versus time under normal growth conditions. A, PET movie frames for plant no. 1 (for the complete movies, see Supporting Information, Movie S-1). The yellow numbers indicate the time elapse since the root supply of 13 NO3-. B, Typical fused PET/CT tomograms from the coronal direction for plant no. 1 at 42 min after 13NO3- was root-supplied (for the complete 35 PET/CT tomograms, see Supporting Information, Figure S-1). The white numbers indicate the tomogram sequence. C, PET movie frames for plant no. 2 (for the complete movie, see Supporting Information, Movie S-2). For A and C, the seven movie frames were captured from the same visual orientation with respect to each individual plant. Color bars indicate the relative radiation intensity to the corresponding subfigures in multicolor.

the plant being “static” rather than “growing”. To address these problems, the culturing pot with the test plant was directly fixed onto the scanner bed and exposed to the PET/CT detectors. To image 13N in the whole plant, the root included, 13NO3- was directly injected into the culturing solution. However, we soon learned that the light emission from the 13NO3- nutrient solution was so strong that the root was undistinguishable from the surrounding solution. In addition, this direct exposure caused severe background noise. To suppress the undesirable noise, the root, along with the 13NO3- nutrient solution, was shielded in a lead pot, as shown in Figure 1. Taking into account the compositional difference between the tissue of animals and plants, CT and PET scan parameters were optimized experimentally for image clarity. The parameters for the CT scan were selected as follows: 140 kV, 10 mA, 5 mm thickness, 512 × 512 matrix, 0.5 s/rev tube rotation speed, HQ (high quality) scan type, 50 cm SFOV (scan field-of-view). The PET acquisition parameters were 4.25 mm thickness, 128 × 128 matrix, 2 iteration, 16 subset, 4.25 mm reconstruction thickness using filtered back projection with a Hanning 8.5 mm smoothing window, smoothing with a Gaussian filter of 8 mm fwhm (full width half-maximum), and 3 min per scanning bed position. The multiring PET scanner used in our experiments operates in both 3D and 2D imaging modes, and both contribute the actual 3D information due to the intrinsic nature of PET methodology. The 3D mode has the advantage of higher sensitivity whereas the 2D mode offers higher signal-to-noise ratio by restricting the scatter and random γ photons.7 By comparing the images obtained using the 2D and 3D modes, 2D-imaging with much lower background and sufficient sensitivity was obtained. A large background in the image reconstructions has been shown to introduce large bias to multiple analysis results.30 Therefore, 2D mode was chosen for the PET scan, and the images were reconstructed using the 2D ordered-subset expectation maximization method. (30) Townsend, D. W.; Isoardi, R. A.; Bendriem, B. In The Theory and Practice of 3D PET; Bendriem, B.; Townsend, D. W., Eds.; Kluwer: Dordrecht, 1998; pp 111-132.

Analysis under Normal Growth Conditions. Under the same temperature and lighting conditions as the normal solution culture, the 13NO3- uptake and 13N distribution versus time in kohlrabi plant no. 1 were examined. Representative results as frames of PET movies and fused PET/CT tomograms are shown in Figure 2A and 2B. The 13NO3- labeling sequences started with the stem close to the root, then the bottom half of the corm, followed by the terminal bud and the corm axillary buds, and last, the petioles. By measuring the duration of 13 NO3- transport from the 13NO3- being root-supplied to the target tissue being labeled and the exterior distance between the root and the target tissue, the 13NO3- transport velocity could be estimated; for example, that from the root to the first labeled petiole was estimated to be 1.0 cm/min. The PET movies (see Supporting Information, Movie S-1) show that 13N was transported through the shell-shaped pathway corresponding to the corm. Interestingly, some strongly labeled routes are visible in the shell-shaped pathway. With close observations of plant no. 1, these strongly labeled routes possess the shortest distances from the corm bottom to corresponding leaves or buds. These observations were verified with additional experiments using other kohlrabi samples. The typical results of these experiments, performed with plant no. 2, are shown in Figure 2C as the frames of a PET movie (see Supporting Information, Movie S-2). The above observations suggest that in kohlrabi, the transport of nitrate-sourced nitrogen is through the shortest route to target tissue. Whether this finding could be extrapolated to other higher plants or to the transport of other molecules warrants further studies. Conducting tissues, such as those of stems or petioles, may provide the best index for the response of plants to nutrients and the nutrient status of plants.19 Close inspection of petioles with13N labeling suggests that the second newest big leaves take precedence over smaller and older ones. Prior to its utilization in amino acid synthesis, nitrate is reduced to nitrite in the cytoplasm by nitrate reductase and nitrite to ammonium by nitrite reductase (NiR) in the chloroplasts/plastids, utilizing the energy and reductants of photosynthesis.19 Prioritized Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

581

Figure 3. The quantification of 13N in terms of SUV versus time analyzed at seven putative sites of interest. The moment of supplying 13NO3to the kohlrabi root was defined as the starting time.

transport of nitrate and nitrite to the second newest big leaves is hypothesized because they have more energy and reductants than the smaller or older ones. PET/CT imaging permits the quantification of a labeled molecule in an interior region of a living body by measuring the SUV at the region. Thus, by measuring SUVs at different regions for one PET/CT imaging, the simultaneous 13N distribution in a growing plant can be described. Similarly, by measuring the SUVs for the same region through multiple imaging, variation of the 13N distribution versus time in the region can be evaluated. For the 13N quantification, seven putative sites of interest (Figure 1), three from the stem (sites 1, 2, and 3) and one each from the corm (site 4), corm axillary bud (site 5), the terminal bud (site 6), and the petiole (site 7), were selected. Their SUVs versus time were measured, and the results are shown in Figure 3. The 13N amounts at sites 1, 2, 3, 4, and 7 approach their plateau at 40 min after the supply of 13NO3-, suggesting the balance between the nitrate uptake and nitrate efflux/utilization in the test plant. As such, the SUVs, measured 40 min after 13 NO3- supply, may represent the relative nitrate-sourced nitrogen amounts (vacuolar nitrate excluded)17,31 at these sites. The amount of 13N at sites 5 and 6 are notable in their continuous increase. This observation is presumably due to the vigorous growth of the terminal bud and the axillary bud, which consumes amino acids, and proteins synthesized in the whole plant; 13N continuously accumulates in these two regions. For the same reason, the terminal bud displays a steeper slope than the axillary bud dose, as shown by Figure 3. Multiple Growth Conditions Analysis. Understanding the physiological and biochemical adjustments caused by environmental changes is a long-term goal in plant research. Enormous efforts have been made by various groups of investigators to study the possible implications of stress conditions for plant growth.20 The capability to analyze the growing plant and to conduct sameplant multiple analysis render PET/CT imaging useful for such studies. In this study, MSX, a potent inhibitor of GS, was selected to induce the stress condition. Disparate conclusions have been drawn regarding nitrate uptake in plants when they are subjected to MSX stress.15,16,32 PEC/CT analysis is also expected to provide (31) Kronzucker, H. J.; Siddiqi, M. Y.; Glass, A. D. M. Planta 1995, 196, 674– 682. (32) Vidmar, J. J.; Zhuo, D.; Siddiqi, M. Y.; Schjoerring, J. K.; Touraine, B.; Glass, A. D. M. Plant Physiol. 2000, 123, 307–318.

582

Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

direct experimental evidence and additional insights into these inconclusive studies. For easy recognition of a leaf in the images, plant no. 3, with only four leaves, was chosen for analysis. 13N was examined in growing kohlrabi no. 3 under normal, MSX stress, and recovery from MSX stress conditions. As the control experiment, plant no. 3 under normal growth conditions was imaged first, and the results are summarized in Figure 4A and 4a, where the main transport of 13N through one-half of the corm is clearly revealed. Close observation found that the four leaves of plant no. 3 were located on almost the same half of the corm. This pattern of nitrate transport is consistent with our observation in plant no. 1, that nitrate is transported through the shortest route to the target tissue. Figure 4B and 4b show the experimental results after plant no. 3 had been treated with MSX via solution culture to its root. When compared with the control experiments, the 13NO3- uptake was significantly reduced and the 13N transport to the leaves was almost undetectable. These observations indicate that although MSX blocked the ammonium-to-glutamine pathway, the conversion of nitrate to ammonium may still proceed and result in ammonium accumulation which tends to cause cell toxicity.33 The plant downregulated its nitrate uptake, as well as the nitrate/nitrite transport to the leaves. Another possible mechanism is that GS activity inhibition resulted in the accumulation of glutamic acid and the plant decreased its nitrate uptake as a feedback response. Our results appear to support the downregulation of nitrate uptake when the kohlrabi was subjected to MSX stress. Plant no. 3 would recover its nitrate metabolism if the MSX stress were removed. To view this recovery, the root of plant no. 3 was washed with fresh nutrient solution to remove the MSX residue once the MSX stress experiment was completed. After plant no. 3 had been cultured under normal conditions for 3 h, the 13NO3- uptake and 13N distribution during the recovery from MSX stress were imaged, and the results are summarized in Figure 4C and 4c. When the results in Figure 4C and 4c are compared with the results in Figure 4B and 4b, plant no. 3 appears to have resumed its nitrate metabolism as a whole. Analogous 13N quantification for some putative sites of interest was carried out. Given that radiation from the petioles and terminal bud under MSX stress and during recovery from (33) Britto, D. T.; Kronzucker, H. J. J. Plant Physiol. 2002, 159, 567–584.

Figure 4. Summary of the PET/CT imaging results of plant no. 3 under multiple growth conditions. A, PET movie frames under normal growth conditions. a, Selected fused PET/CT tomograms taken at 68 min after 13NO3- was root-supplied under normal growth conditions. B, PET movie frames under MSX stress. b, Selected fused PET/CT tomograms taken at 68 min after 13NO3- was root-supplied under MSX stress. C, PET movie frames after the plant had been relieved of MSX stress for 3 h and then root-supplied with 13NO3-. c, Selected fused PET/CT tomograms taken at 68 min after 13NO3- was root-supplied during the recovery from MSX stress. For the A, B, and C complete 3D movies, see Supporting Information, Movie S-3. The yellow numbers indicate the time elapsed since the root supply of 13NO3-. All of the PET/CT tomograms are shown from the coronal direction, and their sequence is numbered in white.

MSX stress was too weak to be measured, only five putative sites of interest (one stem site, three corm sites, and one axillary bud, Figure 5D) were selected for analysis. The SUVs versus time were measured as depicted in Figure 5 under normal, MSX stress, and recovery from MSX stress conditions. The obtained results demonstrate the useful application of PET/CT imaging for quantitative analysis of stress-induced bioresponse. For the nitrate metabolism recovery, whether certain tissues take priority over others is a question of interest. To gain a primary understanding of this question, the recovery rate is defined as follows: recovery rate ) SUVrecovery/SUVnormal - SUVMSX/SUVnormal × 100% 1 - SUVMSX/SUVnormal where the SUVnormal, SUVMSX, and SUVrecovery are the SUVs at certain regions (sites) measured with respect to the subscript conditions. Recovery rate represents the percentages of recovered 13N with respect to the decrease due to MSX stress. In our experiments, the recovery rates of five selected sites, corresponding to 68 min after 13NO3- was root-supplied, were calculated to be 17%, 23%, 30%, 6%, and 24%, respectively. The axillary bud apparently had the slowest 13N recovery. The results in Figure 5A show that the axillary bud exhibited continuous 13N accumulation, and this 13N accumulation implies that the axillary bud was the consumer of amino acids and/or

proteins. Possibly, due to the MSX treatment, plant no. 3 was deficient of amino acids and/or proteins, and, as a result, the transport of amino acids or protein to the axillary bud was greatly downregulated. CONCLUSIONS Three-dimensional PET/CT was applied for the first time to the analysis of nutrient molecules in a growing higher plant. Analysis procedures and operations for solution-cultured growing kohlrabi were established. On the basis of reconstructed PET/ CT tomograms and movies, the uptake processes of 13NO3- were directly visualized, and the variations of 13N distribution in different kohlrabi tissues were quantified. The same-plant multiple PET/CT analysis was exemplified by imaging 13NO3uptake and 13N distribution under normal growth, MSX stress, and recovery from MSX stress conditions. Considering that PET/CT systems for plant imaging are not yet commercially available, efforts to develop analysis procedures and image reconstruction have to be made. In addition, software packages for better attenuation corrections specifically geared toward plant tissue are needed. Although the inherent nature of positron-emitting radioactive isotope labeling restricts the resolution of PET/CT, its high sensitivity and noninvasive capability and its temporally, spatially, and anatomically resolved measurements allow for new insights into research of higher plants. Increasing applications of PET/CT are expected to address various biological problems such as plant nutrition, stress responses, and, in Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

583

Figure 5. Quantification of 13N in terms of SUV versus time for five selected sites corresponding to the following growth conditions: Normal (A), MSX stress (B), and recovery from MSX stress (C). The locations of the five putative sites of interest are indicated in D with numbers.

particular, the topical research of identifying airborne signaling routes of the defense hormone methyl salicylate or methyl jasmonate.34,35 ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant number 20675050). (34) Park, S.-W.; Kaimoyo, E.; Kumar, D.; Mosher, S.; Klessig, D. F. Science 2007, 318, 113–116. (35) Howe1, G. A.; Jander, G. Plant Biol. 2008, 59, 41–66.

584

Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

SUPPORTING INFORMATION AVAILABLE Thirty-five representative PET/CT tomograms from the coronal direction for plant no. 1; 3D PET movies, each from plant no. 1 under normal conditions (exported both from Xeleris and Mimvista), plant no. 2 under normal conditions, and plant no. 3 under normal, MSX stress, and recovery from MSX stress conditions. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 22, 2010. Accepted November 19, 2010. AC102510F