What you can see by developing real-time radioisotope imaging system for plants: from water to element and CO2 gas imaging
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Since plants live on inorganic elements, absorbing ions from roots and transferring them to each tissue in a plant is an essential activity. However, little is known about the movement of the elements or water in plant tissue. Though fluorescent imaging is now overwhelmingly used at the microscopic level in biology, especially to visualize chemicals or organelles in a cell, radioisotope imaging has become one of the important methods for human imaging in the medical field. In the case of plant studies, however, real-time radioisotope imaging is little-known among plant researchers. The author has developed radioisotope imaging systems using various radioisotopes to study living plant activity, both for elements and for water. Here we review the real-time radioisotope imaging methods we developed, and show new aspects of plant physiology discovered by live imaging.
KeywordsReal-time radioisotope imaging system Water movement Element movement Imaging photosynthate movement Carbon fixation Plants
Introduction—Inorganic Elements in Plants 
Water Movement 
It is not known how the difference in the accumulation pattern specific to each element was occurred. If an element is absorbed as an ion dissolved in water, there must be some relationship between water absorption movement and that of the ion. Therefore, we used 15O to label water and measured how water was absorbed and transferred. After supplying the labeled water to the plant we measured the radiation coming from 15O to determine how much of the water was moving inside the plant and was transferred to the targeted stem (1 cm of the internode, just above the root). Because of the extremely short half-life of 15O (i.e., 2 min), no other research group had attempted to use 15O-labeled water to measure the absorption movement in plants. We found that a tremendous amount of water was always leaking out horizontally from xylem tissue in the stem and returning to the xylem again and then moved upward to renew the water at higher position. Since this water circulation in the stem was never reported before, 3H-labeled water was used to confirm the water leakage from xylem tissue and returning movement. In this case the imaging could not be the real-time one, for when 3H-labeled water was applied and moved inside the plant, the beta-ray energy emitted from 3H is too low to detect from outside of the plant. Many plants were prepared, and 5 s after the water was supplied the plant stem was sliced quickly to take an autoradiograph to obtain 3H-water distribution.
Advantage of radioisotope imaging
Radioisotopes have been widely used for tracer work. To get the image, an autoradiography method is employed, where plants were placed on an X-ray film or an imaging plate after application of the radioisotopes to obtain the radioisotope image by radiation exposure. This method provides only a static image, since the plants used for the autoradiography were not able to be used for further experiments.
Because of high sensitivity and the wide range of detection, from trace amount to a large amount of the radioisotopes, autoradiography has come to be used mainly for genetic engineering work; however, this type of the autoradiography has been replaced gradually by fluorescent imaging. Still now, imaging techniques in biology have been quickly developing, especially using fluorescent probes to image chemicals or organic components in cells.
In contrast to fluorescent imaging, there had been no attempt to perform real-time imaging using radioisotopes, except for positron emitters. In the medical field, imaging using positron emitters is well developed and is known as PET (Positron Emission Tomography). In the case of plants, since plants require light, radioisotope imaging has an advantage that it can be conducted either light or dark conditions, whereas fluorescent imaging can be performed only under dark conditions.
PET has become one of the most popular diagnostic tools for patients using the positron emitters with short half-lives, and some research groups have used this kind of imaging technique for plants. However, because of the “positron escape” phenomenon, the intensity of the image can change drastically according to the thickness of the sample, resulting in an image of a stem absent its leaves. The phenomenon also decreases the resolution of the image. As is known, the resolution of the PET images cannot be less than the order of millimeters.
In the case of medical imaging, there are many restrictions for the radioisotopes chosen for human applications: half-lives, radiation energy, etc. However, in the case of plants, to overcome these disadvantages of positron imaging it is preferable to employ other kind of radioisotopes: gamma- or beta-ray emitters, including commercially available ones. The radioisotopes with relatively long half-lives can be the candidates for plants. Since plants require inorganic elements to grow, various kinds of radioisotopes have been used for tracer work to study plant physiology, especially for nutritional work. Therefore, when there is no available radioisotope for the element (for example B, Al, and Si), the study of the element in plant physiology is far behind. Because of this reason, we have been producing 28Mg and 42K ourselves for imaging and tracer works. 28Mg is produced by irradiating an Al target with an alpha beam, followed by chemical separation; and 42K is produced from an 42Ar generator. The half-lives of both elements are short: 21 h and 12 h for 28Mg and 42K, respectively.
Real-Time Radioisotope Images 
The real-time images in Fig. 5 showed that, in the case of phosphate, only a limited amount of the phosphate in soil was absorbed. Since phosphate is liable to be adsorbed in the soil, only the phosphate adsorbed in the vicinity of the root was dissolved by roots for absorption. The images also showed that there was always an accumulation at the root tip where phosphate is required to for proliferation to create new tissues, therefore, sometimes the phosphate moved from upper root to the root tip.
Another moving force for inorganic ions in a plant is photosynthesis. Although the mechanism of photosynthesis is well studied, there is very little information known about the actual movement of the assimilated carbon compounds/metabolites within plants. Therefore, we prepared 14C-labeled carbon dioxide gas and exposed to the plant to it.
Each element has its specific distribution pattern in a plant
Water is circulating in the stem, replacing the water already existing in the tissue with newly absorbed water. In the case of a soybean plant, half of the water already existed in the stem was renewed within about 20 min.
A real-time radioisotope system was developed to visualize the movement of each element, from roots to the above-ground part. Ions absorbed move differently compared to water.
There was a large difference in element uptake manner and growth of the plant between water culture and soil culture.
Carbon fixation and movement of photosynthesis metabolites were able to be visualized. The direction of the photosynthate was different according to the tissue created.
In general, elements necessary for plant growth preferentially move to the youngest tissue, including root tips.
It is interesting to note that the carbon metabolites produced from photosynthesis, which constitute the main part of the plant tissue, move differently according the tissue where metabolites were created. To study all these kinetics, utilization of radioisotopes is the best way. From the results of imaging we can discover new physiological aspects of plants.
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