Abstract
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.
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Introduction—Inorganic Elements in Plants [1]
Plants live on as many as 17 inorganic elements but according to the availability of the elements, they have been developing strategies, such as secreting chemicals or substituting one element with another. Therefore, there are large differences in content of each element among plant species, reflecting different environmental conditions. But what about the concentration difference within plant tissue? Through neutron activation analysis we found that the concentration of each element was drastically different among the tissue of the plant, by factors of 10, 100, 1000, or more. When we analyze the tissue of animals, we do not expect that the concentration of one element in an arm muscle, for example, is 1000 times higher than that in a leg muscle. However, in plants, there is always a concentration gap between different tissues, such as root and above-ground part, internode and leaf stem, for almost all elements. Moreover, there is always an element-specific concentration profile within a plant. For example, heavy elements tend to accumulate in roots. Ca and Mg accumulate during the young age around the cotyledon, and when upper tissue began to develop they moved upward (Fig. 1). Even in one leaf or in one internode, there was a gradient of element concentration. The element profile pattern within a plant was kept in the same manner, throughout the developmental stage. However, in the case of volatile elements such as Cl and Br, some of the elements distributed in a plant were lost from leaves and the content decreased during growth.
Water Movement [2]
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.
In the case of 15O-water, the water absorption curve was only able to measure until 1200 s after application because of the short half-life of 15O. Through simulation it was found that within 20 min about half of the water already existed in the stem was replaced by newly absorbed water (Fig. 2). Water movement we could measure was so different from the movement of dissolved ions that, we wanted to image the real-time element absorption movement.
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.
Development of Real-Time Radioisotope Imaging System [3,4,5,6,7,8,9]
We targeted conventional gamma- or beta-ray emitters and we have been developing the real-time imaging systems for macroscopic and microscopic samples. A schematic illustration of the real-time imaging system is shown in Fig. 3. The radiation emitted from the plant after application of the radioisotopes was converted to light by a scintillator deposited on a FOS (Fiber Optic Plate) and the light image taken by a highly sensitive CCD camera. After evaluation of several types or thickness of the scintillator, now CsI is used. To image an entire plant from root to shoot, the macroscopic imaging system is used, and for microscopic imaging a modified fluorescence microscope is used to obtain three images simultaneously: light, fluorescent, and radioisotope images. We now can image the following 15 elements, 14C, 18F, 22Na, 28Mg, 32P 33P, 35S, 42K, 45Ca, 48V, 54Mn, 55Fe, 59Fe, 65Zn, 86Rb, 109Cd, and 137Cs.
Real-Time Radioisotope Images [10]
The images obtained to date have shown that the moving speed from root to the highest part of the plant is very element specific. The moving speed of the heavy elements from roots to the tip of the leaves is faster than the other elements, whereas Mg or Ca move very slow (Fig. 4).
When the plants growing in soil and those in water culture solution were compared, plants grew faster in water culture because of the easier acquisition of elements dissolved in ionic forms in water (Fig. 5), however, the yield of the seeds is low. It seems that the plants growing in water culture do not have the same necessity to produce a large number of seeds to create the next generation, since the nutritional environment is so rich, with plenty of nutrition in water culture. Usually, when the nutritional condition is poor, plants create a large amount of seeds for the next generation to raise the possibility of the survival rate of the species.
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.
Visualization of Photosynthesis [11,12,13,14]
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.
The carbon fixed by photosynthesis creates almost all the tissue of the plants. That means that plants are always creating visible tissue out of invisible carbon dioxide gas in air. It was so impressive to visualize how the carbon dioxide gas in the air was fixed at the above-ground plant and the assimilated carbon metabolites are transferred to create new tissue, for example, new root tip (Fig. 6).
We could show the different direction of the photosynthates movement according to the 14C-gas fixation site. In the case of Arabidopsis, carbon fixed at the site of the rosette (large leaves spread out in a circle just above the root) is transferred mainly to the main stem tip and roots, whereas the carbon fixed in the small leaves or stems were moved mainly to the tip of a branched stem and does not reach the root (Fig. 7). In the soybean pod, the carbon metabolite from photosynthesis was mainly supplied from the closest leaves to the pod.
Summary
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1.
Each element has its specific distribution pattern in a plant
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2.
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.
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3.
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.
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4.
There was a large difference in element uptake manner and growth of the plant between water culture and soil culture.
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5.
Carbon fixation and movement of photosynthesis metabolites were able to be visualized. The direction of the photosynthate was different according to the tissue created.
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6.
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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Nakanishi, T.M. What you can see by developing real-time radioisotope imaging system for plants: from water to element and CO2 gas imaging. J Radioanal Nucl Chem 318, 1689–1695 (2018). https://doi.org/10.1007/s10967-018-6324-0
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DOI: https://doi.org/10.1007/s10967-018-6324-0