Water-Specific Imaging

Our first target was water, namely, how to obtain a water-specific image nondestructively. Using a neutron beam, we could visualize water-specific images of plants, including roots and flowers, which were never shown before. Each image suggested the plant-specific activity related to water.We briefly present how to acquire the image and what kind of water image is taken by neutron beam irradiation. We present a variety of plant samples, such as flowers, seeds, and wood disks. It was noted that neutrons could visualize the roots imbedded in soil without uprooting. When a spatial image of the root imbedded in soil was created from many projection images, the water profile around the root was analyzed. Then, fundamental questions were raised, such as whether plants are absorbing water solution or water vapor from the soil, because there was always a space adjacent to the root surface and hardly any water solution was visualized there. The roots are in constant motion during growth, known as circumnutation, and it is natural that the root tip is always pushing the soil aside to produce space for the root to grow. If the roots are absorbing water vapor, then the next question is about metals. Are the roots absorbing metal vapor? Since we tended to employ water culture to study the physiological activity of plants, the physiological study of the plants growing in soil was somewhat neglected. Later, when we could develop a system to visualize the movement of element absorption in a plant, there was a clear difference in element absorption between water culture and soil culture.

Real-Time Element Movement in a Plant

We developed an imaging method utilizing the available RIs. We developed two types of real-time RI imaging systems (RRIS), one for macroscopic imaging and the other for microscopic imaging. The principle of visualization was the same, converting the radiation to light by a Cs(Tl)I scintillator deposited on a fiber optic plate (FOS). Many nuclides were employed, including 14C, 18F, 22Na, 28Mg, 32P 33P, 35S, 42K, 45Ca, 48V, 54Mn, 55Fe, 59Fe, 65Zn, 86Rb, 109Cd, and 137Cs.Since radiation can penetrate the soil as well as water, the difference between soil culture and water culture was visualized. 137Cs was hardly absorbed by rice roots growing in soil, whereas water culture showed high absorption, which could provide some reassurance after the Fukushima Nuclear Accident and could indicate an important role of soil in firmly adsorbing the radioactive cesium.28Mg and 42K, whose production methods were presented, were applied for RRIS to visualize the absorption image from the roots. In addition to 28Mg and 42K, many nuclides were applied to image absorption in the roots. Each element showed a specific absorption speed and accumulation pattern. The image analysis of the absorption of Mg is presented as an example. Through successive images of the element absorption, phloem flow in the aboveground part of the plant was analyzed. The element absorption was visualized not only in the roots but also in the leaves, a basic study of foliar fertilization.In the case of the microscopic imaging system, a fluorescence microscope was modified to acquire three images at the same time: a light image, fluorescent image, and radiation image. Although the resolution of the image was estimated to be approximately 50 μm, superposition showed the expression site of the transporter gene and the actual 32P-phosphate absorption site to be the same in Arabidopsis roots.

Other Real-Time Movement

In the case of root movement, a very interesting phenomenon was found using the Super-HARP camera, which enabled the visualization of root movement in the dark.Although the first data on plants show that the harmful effect is growth inhibition, the first effect of the toxicity was to stop the rotation movement of the roots before growth inhibition occurred.When there was a chemical change in the environment, although the circumnutation of the root tip ceased, the root was able to elongate, and it was interesting that after a while the root movement resumed. In the case of a rice root, one round of movement of the rice root tip showed a constant time of approximately 50 minutes. However, this movement ceased when Al ion was supplied. The time needed for resuming the movement of the root tip was dependent on the Al ion concentration. It is not known what triggers the resumption of the movement of the root tip.

Real-Time Water Movement in a Plant

The next approach to research on water was to measure the small amount of water actually moving within a plant. The best method is to utilize radioisotope (RI)-labeled water and measure the radiation from outside of the plant. However, it is rather difficult to label water, since there are only limited kinds of RI for tracing water.When utilizing 18F to trace water movement, another fundamental question to consider was the features that characterize drought-tolerant and drought-sensitive plants. It is natural to suppose that drought-tolerant plants have strong water absorption; therefore, by analyzing the water absorption mechanism of tolerant plants and by introducing this function to sensitive plants, it might be possible to make the sensitive plants more tolerant.However, when water uptake was studied in naturally developed drought-tolerant and drought-sensitive cowpea, selected from 2000 cowpea plants grown in the field of Africa, the result was unexpected. Under normal conditions, the amount of water absorbed by the drought-tolerant strain was much lower than that absorbed by the sensitive strain, as if showing the low capability of water absorption. When a drought condition was introduced, the tolerant strain began to absorb much more water than usual, whereas the sensitive strain could not absorb as much water as before. This result provided us with an important lesson. Analyzing the mechanism of drought tolerance only by comparing the water absorption of tolerant and sensitive plants might not readily reveal the reason for drought tolerance. The features of the naturally produced plants showed us different mechanisms that might not match our expectations developed in the laboratory.Next, we performed water measurements using 15O-labeled water, which has an extremely short half-life of 2 minutes. Here, we found another astonishing result, which was “water circulation” in the plant internode. A tremendous amount of water was always leaking from xylem cells, which had been regarded as a mere pipe to transfer water from the root to the aboveground parts. In another subsequent study, it was shown that the water flowing out from the xylem was pushing out the water already present in the stem and then returning to the xylem again to move upward. The water velocity in the internode was kept constant, and through simulation, it took less than 20 minutes to exchange the water already present in the stem with newly absorbed water.

Microautoradiography (MAR)

MAR was developed to increase the resolution of the image for the sliced plant sample. This revised MAR method showed the detailed distribution of 137Cs accumulation in embryos, which indicated that the plumule and radicle, which grow as a meristem of a root or shoot, were protected from the accumulation of heavy elements. Because of daguerreotype imaging, MAR is now hardly used, and the film emulsion is not available. Here, this method was essentially recreated with a revised processing method for both sample preparation and imaging process.

Visualization of 14C-labeled Gas Fixation in a Plant

We targeted not only the elements we can supply to the nutrient solution but also carbon dioxide gas to visualize the fixation process and the movement of assimilated carbon in a plant. This is another highlight of our study using real-time RI imaging systems (RRIS). The interesting result was that the route of assimilated carbon was different depending on where the fixation took place. In Arabidopsis, most of the metabolites after photosynthesis were transferred to the tip of the main internode and roots when 14CO2 gas was fixed and photosynthates were produced at rosette leaves, whereas most of the metabolites moved to the tip of the branch internode and hardly moved down to the roots when 14CO2 gas was supplied to the aboveground parts of the plant other than rosette leaves. Interestingly, it was possible to visualize and trace which tissue performed the fixation of 14CO2 gas, i.e., carbon could be traced from the fixation site in tissue to tissue formation. However, especially in the case of 14C imaging, image analysis should be carefully performed because of the self-absorption of the β-rays in tissue. To image 14CO2 gas fixation in larger samples, approximately 50 cm in height, a plastic scintillator was introduced, and the assimilation process of the gas was visualized for rice and maize.