Ryohei Sugita

Evaluation of in vivo detection properties of 22Na, 65Zn, 86Rb, 109Cd and 137Cs in plant tissues using real-time radioisotope imaging system.

In plant research, radioisotope imaging provides useful information about physiological activities in various tissues and elemental transport between plant organs. To expand the usage of imaging techniques, a new system was developed to visualize beta particles, x-rays and gamma-rays emitted from plant bodies. This real-time radioisotope imaging system (RRIS) visualizes radioactivity after conversion into light with a CsI(Tl) scintillator plate. Herein, the RRIS detection properties of the gamma-ray emitters (22)Na, (65)Zn, (86)Rb, (109)Cd and (137)Cs were evaluated in comparison with those of radioluminography (RLG) using an imaging plate. The lower quantitative detection limit (Bq mm(-2)) during a 15 min period ranged from 0.1 to 4, depending on the nuclide, similar to that of RLG. When the quantitative ability to detect radiation from various Arabidopsis tissues was analyzed, the quantitative capability in silique and the thick internode tended to be low. In an EGS5 simulation, beta particles were the greatest contributors to RRIS imaging of (22)Na, (86)Rb and (137)Cs, and low-energy x-rays contributed significantly to (65)Zn and (109)Cd detection. Thus, both self-absorption and air space between the sample and scintillator surface could impair quantitative RRIS imaging. Despite these issues, RRIS is suggested for quantitative time-course measurements of radionuclide motion within plants.

Tracer experiment using 42K+ and 137Cs+ revealed the different transport rates of potassium and caesium within rice roots

The differences in the transport characteristics in planta between potassium (K+) and caesium (Cs+) was investigated using their radionuclides, 42K+ and 137Cs+. A tracer experiment using nutrient solutions supplemented with 42K and 137Cs revealed that the ratio of the roots K+ uptake rate to its Cs+ uptake rate was 7-11 times higher than the K+:Cs+ concentration ratio in the solution, and the number was varied depending on the K concentration in the solution and also on the growth condition. After entering through the root tissues, the 42K+:137Cs+ ratio in the shoots was 4.28 times higher than the value in the roots. However, the 42K+:137Cs+ ratio in each leaf did not differ significantly, indicating that the primary transport of K+ and Cs+ in the shoots are similarly regulated. In contrast, among the radionuclides stored in the roots over 4h, 30% of the 42K+ was exported from the roots over the following hour, whereas only 8% of 137Cs+ was exported. In addition, within the xylem, K+ was shown to travel slowly, whereas Cs+ passed quickly through the roots into the shoots. In conclusion, our study demonstrated very different transport patterns for the two ions in the root tissues.

Development of a 14 C detectable real-time radioisotope imaging system for plants under intermittent light environment

A new real-time radioisotope imaging system (RRIS) to study the kinetics of nutrient uptake and transfer of photosynthetic products in a living plant was developed and evaluated through a test run. 14C is a common radioisotope of carbon and useful to trace the photosynthetic products as well as a low energy beta emitter. The rationale of this study was to develop a RRIS that has the ability to detect low energy beta emitters, such as 14C, 35S, and 45Ca. To achieve compatibility between the detection of low energy beta emitters and irradiation of the test plant, an intermittent lighting system was added to the RRIS. Furthermore, a commercially available digital camera was added to the RRIS for acquisition of photographic images of the test plants. The capabilities of the new RRIS were evaluated through a test run by using seedlings of rice plants and 35S-labeled sulfate. It was shown that the new RRIS was able to detect 35S absorbed by rice plant seedlings, and it was able to acquire photon-counting images and photographic images of the test plants simultaneously. Despite some limitations, the new RRIS provides a means to study the kinetics of elements in plants by utilizing low energy beta emitters.

Nondestructive real-time radioisotope imaging system for visualizing 14C-labeled chemicals supplied as CO2 in plants using Arabidopsis thaliana

We have developed a real-time radioisotope imaging system (RRIS) that can nondestructively trace 14C-labeled chemicals in plants. In an experiment, after feeding 14CO2 to a plant, the plant was fixed inside a box where lighting was regulated, and beta rays emitted from the 14C in the plant were intermittently imaged using the developed system. As a first step, using a series of standard sources of 14C, the data depth and detection limits of the 14C images captured by the RRIS were evaluated for various integral times. As a result, the linearity between the 14C activity and signal intensity was determined for the range 103. Next, the linearity was validated using plant (Arabidopsis thaliana) organs, resulting that the linearity was shown in the case of young leaf, but was not maintained in the thick organs, such as a flower, mature leaf, siliques, and stem. Considering the good correlation between the intensity by RRIS and the PSL value by an imaging plate (IP) as well as the relative low energy of beta rays emitted from 14C, the thickness of the organs would easily affect the quantitativity of the RRIS as well as an IP. Our findings prove that sequential images of 14C in a living plant sample in a regulated light and air environment can be nondestructively analyzed using the developed system, whose quantitativity is similar to that of an IP.

Visualization of Uptake of Mineral Elements and the Dynamics of Photosynthates in Arabidopsis by a Newly Developed Real-Time Radioisotope Imaging System (RRIS)

Minerals and photosynthates are essential for many plant processes, but their imaging in live plants is difficult. We have developed a method for their live imaging in Arabidopsis using a real-time radioisotope imaging system. When each radioisotope, 22Na, 28Mg, 32P-phosphate, 35S-sulfate, 42K, 45Ca, 54Mn and 137Cs, was employed as an ion tracer, ion movement from root to shoot over 24 h was clearly observed. The movements of 22Na, 42K, 32P, 35S and 137Cs were fast so that they spread to the tip of stems. In contrast, high accumulation of 28Mg, 45Ca and 54Mn was found in the basal part of the main stem. Based on this time-course analysis, the velocity of ion movement in the main stem was calculated, and found to be fastest for S and K among the ions we tested in this study. Furthermore, application of a heat-girdling treatment allowed determination of individual ion movement via xylem flow alone, excluding phloem flow, within the main stem of 43-day-old Arabidopsis inflorescences. We also successfully developed a new system for visualizing photosynthates using labeled carbon dioxide, 14CO2. Using this system, the switching of source/sink organs and phloem flow direction could be monitored in parts of whole shoots and over time. In roots, 14C photosynthates accumulated intensively in the growing root tip area, 200–800 µm behind the meristem. These results show that this real-time radioisotope imaging system allows visualization of many nuclides over a long time-course and thus constitutes a powerful tool for the analysis of various physiological phenomena.

Carbon-14 labelled sucrose transportation in an Arabidopsis thaliana using an imaging plate and real time imaging system

As an approach to increased production of rape seed oil from Brassica napus L., Arabidopsis thaliana, a species from the same Brassicaceae family, was used to investigate transport behavior and distribution of matter in the plant body. In this study, sucrose, an initial metabolic product of photosynthesis, labeled with carbon-14 was used. The sucrose was applied to A. thaliana via the surface of a rosette leaf. Using the real time radioisotope imaging system we developed and an imaging plate (IP), images of whole or part of the sample were obtained. The sucrose assimilation products were accumulated in maturing tissue such as flowers and fruits, and in a joint part. From the comparison among branches and stems, it was indicated that there were different patterns of demand and distribution of sucrose assimilation products depending on the tissue and its growing stage. This might be caused by either morphological reason such as diameter and location of the sieve tube, or genetic factors such as an activity of a membrane transport protein. Because of self-absorption of carpels, it was difficult to observe the accumulation of carbon-14 in the seeds inside the fruits; however, an IP image of a frozen section of a fruit revealed that carbon-14 transport to seeds was higher than that of carpels. These methods will help us gain insight into matter transport and strategies to improve the production of rape seed oil.

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.

Imaging Techniques for Radiocesium in Soil and Plants

Various radioisotope imaging techniques have been used at the Graduate School of Agricultural and Life Sciences, University of Tokyo, to analyze samples containing radiocesium (137Cs and 134Cs). There are two types of samples: (1) environmental samples contaminated by the fallout from the Fukushima Daiichi nuclear power plant accident, which contain relatively low concentrations of radiocesium and (2) laboratory samples from tracer experiments conducted at the radioisotope institution containing relatively high concentrations of 137Cs. The first technique used to visualize radiocesium in soil and plants was radioluminography (RLG). RLG, which makes use of an imaging plate, has a dynamic range that is large enough to detect both environmental and tracer-added samples. To quantify radiocesium distributions, the samples were frozen and sliced before contact with the imaging plate. This freezing procedure after sampling is for preventing radiocesium movement during slicing and measurement of 137Cs distribution. After slicing, two detection methods were employed: RLG and microautoradiography (MAR). MAR is the conventional and older method for imaging radioisotopes based on the daguerreotype process. We applied this method to frozen sections and obtained 137Cs distributions at a higher resolution than with RLG. Following this, we employed a non-destructive method for imaging 137Cs movement in a living plant. We developed the visualization technique called real-time radioisotope imaging system and then demonstrated 137Cs movement from soil to rice plants using a chamber containing paddy soil, water, and rice plants. Lastly, 42K obtained by 42Ar–42K generation enabled a comparison between the movement of 137Cs and 42K. The mechanism of Cs transport has been reported to have some relationship with the K transport system, so experiments using both 137Cs and 42K would be useful for clarifying the mechanism in more detail.

Magnesium deficiency damages the youngest mature leaf in rice through tissue-specific iron toxicity

AimsMagnesium deficiency can cause starch accumulation, photosynthesis inhibition and senescence particularly in young mature leaves. This study was performed to identify the initial process leading to leaf senescence under Mg deficiency.MethodsGene expression in the young leaf was analyzed at days 2, 4, 5 of Mg deficiency using microarray analysis, and several Fe responsive genes were identified. Therefore, the effect of lowering Fe supply on gene expression and oxidative stress under Mg deficiency was evaluated.ResultsTranscriptome analysis revealed that 7 of the 30 most upregulated genes and 11 of the 30 most downregulated genes were Fe-responsive. Particularly, the upregulation of OsFER2 and downregulation of OsMIR and OsIRO2 hinted at the induction of excess Fe stress under Mg deficiency. Both lowering of Fe concentration in Mg-free solutions and resupply of Mg without modifying Fe concentrations at day 4 rescued leaves from senescence by inhibiting oxidative stress and normalising the expression of Fe-responsive genes. Meanwhile, Fe content was equal between control, Mg-deficient and Mg-resupplied plants.ConclusionMg shortage can induce excess Fe stress, which in turn causes oxidative stress before inhibition of photosynthesis. It is proposed that Mg deficiency disrupts a mechanism for storing toxic Fe ions into the vacuole in the expanding young leaf cells.