Visualization of zinc dynamics in intact plants using positron imaging of commercially available 65Zn

Background Positron imaging can be used to non-destructively visualize the dynamics of a positron-emitting radionuclide in vivo, and is therefore a tool for understanding the mechanisms of nutrient transport in intact plants. The transport of zinc, which is one of the most important nutrient elements for plants, has so far been visualized by positron imaging using 62Zn (half-life: 9.2 h), which is manufactured in the limited number of facilities that have a cyclotron. In contrast, the positron-emitting radionuclide 65Zn (half-life: 244 days) is commercially available worldwide. In this study, we examined the possibility of conducting positron imaging of zinc in intact plants using 65Zn. Results By administering 65Zn and imaging over a long time, clear serial images of 65Zn distributions from the root to the panicle of dwarf rice plants were successfully obtained. Conclusions Non-destructive visualization of zinc dynamics in plants was achieved using commercially available 65Zn and a positron imaging system, demonstrating that zinc dynamics can be visualized even in facilities without a cyclotron.


Background
Positron imaging is widely used to non-destructively visualize the dynamics of positron-emitting radionuclides in vivo. The most common use of positron imaging is cancer screening by positron emission tomography (PET), which exploits the tendency of fluorodeoxyglucose labeled with the positron-emitting radionuclide 18 F to accumulate in cancer cells. In addition, PET has been used in medical research to analyze the kinetics of drugs labeled with 11 C, 13 N, and 15 O. Meanwhile, in recent years, positron imaging has been used to study plants in research facilities around the world [1][2][3][4][5][6][7][8]. A recent review article emphasized the advantages of positron imaging for understanding the function of the xylem and phloem [9].
Because the major and minor essential elements for plants have a number of positron-emitting radionuclides, positron imaging is a potentially useful tool for understanding the mechanisms of nutrient transport in plants. The radionuclides typically used for positron imaging of plants are limited to 11 C, 13 N, 15 O, and 18 F, which are processed using well-established purification methods developed for medical research [3,9]. We have previously studied positron imaging of minor essential and toxic elements, such as 64 Cu (half-life: 12.7 h) [10] and 107 Cd (halflife: 6.5 h) [11][12][13][14][15][16], using the positron-emitting tracer imaging system (PETIS), a two-dimensional positron imaging system (special resolution: approximately 2 mm). However, this approach involves difficulties in purifying the positron-emitting metal radionuclides in other research facilities. Furthermore, most of these positronemitting nuclides have short half-lives and are therefore not commercially available. Thus, the implementation of positron imaging for plant research is limited to research facilities with a cyclotron, where positron-emitting

Open Access
Plant Methods *Correspondence: suzui.nobuo@qst.go.jp 3 Takasaki Advanced Radiation Research Institute, National Institutes for Quantum and Radiological Science and Technology, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan Full list of author information is available at the end of the article radionuclides can be produced through nuclear reactions using an accelerated ion beam.
Zinc is one of the essential elements for all living organisms, including higher plants. Zinc deficiency in crops is one of the most serious problems in food production worldwide [17]. Therefore, it is important to understand how plants regulate zinc transport. In the efforts to elucidate the mechanism of zinc transport, positron imaging of zinc dynamics has been a powerful tool. The positronemitting radionuclide 62 Zn (half-life: 9.2 h) has been used to study the dynamics of zinc in intact plants [18,19]. 62 Zn decays by electron capture (91.6%) and positron emission (8.4%) and produces the radionuclide 62 Cu (half-life: 9.7 min). Although the positron emission rate of 62 Zn is weak, its daughter 62 Cu decays with a 97.8% rate by positron emission to stable 62 Ni; therefore, a positron imaging system can obtain images of 62 Zn with high efficiency. However, because 62 Cu may migrate differently from 62 Zn in a plant body, there has been the argument that the positron imaging using 62 Zn correctly reflects zinc dynamics in plants. Furthermore, zinc imaging using 62 Zn can only be conducted in facilities with a cyclotron. In contrast, 65 Zn (half-life: 244 days) is commercially available, and is therefore frequently used as a zinc tracer in the field of plant science [20]. 65 Zn decays with a 98.6% probability by electron capture and 1.4% by positron emission to stable 65 Cu. Because the positron emission rate of 65 Zn is too low, 65 Zn has been considered to be unsuitable for positron imaging; however, there has been no direct verification of this claim. Therefore, in this study, we examined whether 65 Zn can be used to visualize zinc dynamics in an intact plant using a positron imaging system, and to estimate the kinetics of zinc uptake using positron imaging data.

Plant cultivation
To visualize zinc dynamics in the whole plant body, dwarf rice plants were cultivated. Seeds of the dwarf rice cultivar Oryza sativa L. cv Waito-C were germinated and each plant was grown in a vinyl pot (12 cm in diameter, 10 cm height) filled with artificial soil (Bonsol; Sumitomo Chemical Co., Tokyo, Japan) in a greenhouse (15- Additionally, typical rice plants (Oryza sativa L. cv Nipponbare) were cultivated hydroponically for four weeks in the modified Kimura B solution and used to evaluate uptake kinetics.

Whole-plant imaging experiment
For whole-plant imaging, the roots of an intact dwarf rice plant were inserted in a 30 mL plastic disposable syringe (Termo Co., Tokyo, Japan), and the shoots were fixed to an acrylic board. The acrylic board, which held two plants at a time, was placed in the field of view of the PETIS (a modified PPIS-4800 positron imaging system; Hamamatsu Photonics, Hamamatsu, Japan) (Fig. 1a). Each syringe was supplied with 30 mL of 0.5 mM CaCl 2 containing 0.1 µM ZnSO 4 (3 nmol in total) labeled with 444 kBq of 65 Zn (22 pmol), which was purchased from RIKEN (Wako, Japan), and 2 mM 2-(N-morpholino)ethanesulfonic acid. The movement of 65 Zn in the belowground parts of the plants, including the roots and the shoot base, was monitored by the PETIS every 10 min for 24 h. Then, the PETIS was moved to the top of the plants and the 65 Zn movement in the above-ground parts was monitored every 10 min for 72 h. In the first 48 h of the imaging experiment (which lasted 96 h in total), the roots were fed with 0.5 mM CaCl 2 containing 65 Zn (the feeding step) and in the last 48 h, they were transferred into Kimura B solution (the chasing step). The solutions were maintained at the same level by supplying 0.5 mM CaCl 2 or Kimura B solution, which does not contain Zn + with the siphon method, as described by Fujimaki et al. [11]. All imaging experiments were conducted in a growth chamber with continuous light at a density of 400 μmol m −2 s −1 .
The time course data of the zinc amount (mol) in the regions of interest in the images were calculated by the values of the signal intensity (cps) extracted using the NIH Image J 1.50 software (http://rsb.info.nih.gov/ij/), counting efficiency of the system (cps Bq −1 ) and specific radioactivity (Bq mol −1 ).

Evaluation of uptake kinetics
To experimentally determine the uptake kinetics of zinc, the roots of six non-dwarf rice plants were inserted in a transparent acrylic root box specialized for direct root imaging [12] (Fig. 3a). Each compartment of the root box was supplied with 14 mL of 0.5 mM CaCl 2 containing different concentrations (0.1, 0.25, 0.5, 1, 2.5 and 5 µM) of ZnSO 4 , labeled with 124 kBq (6 pmol) of 65 Zn and the 65 Zn movement from the solution to the roots was monitored by PETIS for 7 h. The solution was continuously stirred with gentle aeration in order to maintain a uniform composition in each compartment of the root box. The uptake velocities for the various zinc concentrations were calculated every hour using the imaging data acquired from the individual rice plants. The obtained substrate velocity data were fitted with the modified Michaelis-Menten equation according to Claassen and Barber [21]: where, In, c and E are the net uptake velocity, substrate concentration and the efflux velocity, respectively. Vmax, Km and E value were estimated by least-square regression using the python SciPy library (http://www.scipy. org/).

Results
The tracer solution containing 65 Zn was administered to dwarf rice plants and the dynamics of 65 Zn in intact whole plants was monitored by PETIS (Fig. 1a). As a result, obvious clear serial images of 65 Zn distributions from the root to the panicle were successfully obtained for 96 h (Fig. 1b, c). The counting efficiency of PETIS for 65 Zn was 6.68 × 10 −4 cps Bq −1 , which was sufficient to obtain clear serial images of 65 Zn distributions from the root to the panicle. Because the amount of non-radioactive zinc labeled with 65 Zn was calculated by 65 Zn radioactivity, Fig. 2 represents the time course of "newly acquired" zinc. The amount of zinc in the hydroponic solution decreased rapidly for the first 3 h and more slowly after 6 h (Fig. 2b). In contrast, the amount of newly acquired zinc in the root increased for the first 3 h and then decreased for the remainder of the 24 h (Fig. 2c). Because the shoot base and the node were in the field of view of PETIS during the entire measurement (Fig. 2a, e) time course data could be obtained in these regions for 96 h (Fig. 2d, f ). The results show that the amount of newly acquired zinc in the shoot base and the node increased for 48 and 72 h, respectively, and then became plateau (Fig. 2d, f ). Although 65 Zn movement in the panicle was not monitored for the first 24 h, the x-intercept and slope of the linear approximation line fitted to the time course data indicate that zinc was transported from the root and arrived at the panicle 5.3 h after feeding, and monotonically accumulated in the panicle for 96 h at a rate of 5.1 pmol h −1 (Fig. 2g).
We next obtained serial images and quantified zinc uptake in non-dwarf plants for various initial zinc concentrations (0.1-5 µM; Fig. 3b, c). The corresponding zinc uptake velocities were calculated from the decrease in zinc observed in the solution every hour (six values from each plant). The velocity data from the plant fed with 5 µM of initial zinc concentration were excluded from the analysis because the plant was injured during the experimental procedure and the zinc concentration did not monotonically decrease. The obtained substrate

Discussion
In this study, we demonstrated that 65 Zn can be used for positron imaging in plants by administering adequate amounts of 65 Zn and imaging for a sufficiently long time (Fig. 1). In addition, it was demonstrated that the main gamma ray of 65 Zn (1.1 MeV; 50.6%) does not become noise for positron imaging. These results indicate that non-destructive imaging of zinc dynamics can be conducted even in research facilities without a cyclotron. In order to capture 65 Zn movement from the root to the panicle with the restricted size of PETIS field of view, the tested rice plants were artificially dwarfed by treatment with 0.1 ppm of uniconazole Izumi et al. [22] reported that treatment with uniconazole P reduces the fresh weight of both shoots and roots by 39 and 22%, respectively. This result implies that shoot/root ratio is not drastically changed by treatment with uniconazole P. Hence, we sought that the 65 Zn movement in the dwarf rice plant provides qualitative information on the zinc dynamics in normal rice plant. Our time course measurements revealed that after peaking at approximately 3 h, the amount of newly acquired zinc in the root of the dwarf rice plants decreased (Fig. 2c). On the other hand, in a similar experiment using 107 Cd, the amount of cadmium in the root of a japonica rice cultivar plateaued within 1 h and increased slightly but did not decrease [12]. Although zinc and cadmium are considered to partially share the same pathway from root to shoot [23,24], these results suggest that a rice plant usually retains toxic cadmium in the root, whereas it transports zinc to the shoot continuously. The amount of newly acquired zinc in the shoot base became plateau at the end of the feeding step (Fig. 2d), but those in the node and the panicle increased during the chasing step (Fig. 2f, g). These results indicate that zinc absorbed by the root is not accumulated in, but passes through the shoot base to the node and is continuously transported to the panicle in the In addition, we did not observe any 65 Zn signals in the region of the leaf blade (Fig. 1c). Because zinc is considered to transfer from the xylem to the phloem at nodes and finally accumulate in the panicle [25], these results suggest that the positron imaging of 65 Zn correctly captures the zinc translocation in rice plants.
Our zinc uptake evaluation results obtained an estimated Km value of 1.1 µM, whereas the previously reported value was 5.5 µM [26]. The difference may be caused by the lack of zinc deficient treatment before measurement and by the lack of velocity data at higher substrate concentrations than 2.5 µM in this study. Therefore, our estimation of Km using positron imaging data has room for improvement. This approach has the advantage of obtaining several substrate-velocity data measurements from one plant without continuous sampling. Principally, the uptake kinetics of a nutrient element can be estimated from the depletion curve of one plant [21]. The development of this analytical method makes it possible to estimate the Km value from an individual plant.

Conclusions
To the best of our knowledge, this is the first report to visualize zinc movement in living plants using commercially available 65 Zn and a positron imaging system. Thus far, short-lived 62 Zn has been used in developing a cancer screening agent for PET [27]. Although 65 Zn cannot be administered to humans because of its long half-life, it could be applicable to the kinetic analysis of drugs in small experimental animals if close attention is paid to avoid radioactive contamination in laboratories. Therefore, positron imaging of 65 Zn can be useful not only for the study of zinc dynamics in plants but also for the study of the kinetics of drugs labeled with 65 Zn in animals.

Authors' contributions
NS conceived and designed research. YY, SI, HS and NK conducted experiments. NS analyzed data and wrote the manuscript. All authors read and approved the final manuscript.