Satomi Ishii

Real-time imaging and analysis of differences in cadmium dynamics in rice cultivars (Oryza sativa) using positron-emitting107Cd tracer

BackgroundRice is a major source of dietary intake of cadmium (Cd) for populations that consume rice as a staple food. Understanding how Cd is transported into grains through the whole plant body is necessary for reducing rice Cd concentrations to the lowest levels possible, to reduce the associated health risks. In this study, we have visualized and quantitatively analysed the real-time Cd dynamics from roots to grains in typical rice cultivars that differed in grain Cd concentrations. We used positron-emitting107Cd tracer and an innovative imaging technique, the positron-emitting tracer imaging system (PETIS). In particular, a new method for direct and real-time visualization of the Cd uptake by the roots in the culture was first realized in this work.ResultsImaging and quantitative analyses revealed the different patterns in time-varying curves of Cd amounts in the roots of rice cultivars tested. Three low-Cd accumulating cultivars (japonica type) showed rapid saturation curves, whereas three high-Cd accumulating cultivars (indica type) were characterized by curves with a peak within 30 min after107Cd supplementation, and a subsequent steep decrease resulting in maintenance of lower Cd concentrations in their roots. This difference in Cd dynamics may be attributable to OsHMA3 transporter protein, which was recently shown to be involved in Cd storage in root vacuoles and not functional in the high-Cd accumulating cultivars. Moreover, the PETIS analyses revealed that the high-Cd accumulating cultivars were characterized by rapid and abundant Cd transfer to the shoots from the roots, a faster transport velocity of Cd to the panicles, and Cd accumulation at high levels in their panicles, passing through the nodal portions of the stems where the highest Cd intensities were observed.ConclusionsThis is the first successful visualization and quantification of the differences in whole-body Cd transport from the roots to the grains of intact plants within rice cultivars that differ in grain Cd concentrations, by using PETIS, a real-time imaging method.

Kinetic Analysis of Carbon-11-Labeled Carbon Dioxide for Studying Photosynthesis in a Leaf Using Positron Emitting Tracer Imaging System

The positron emitting tracer imaging system (PETIS) and carbon-11-labeled carbon dioxide (11CO2) can be used for imaging the photosynthesis process in plant leaves. Further, 11C kinetics facilitate the estimation of the physiological function parameters of photosynthesis. PETIS measurements were performed under four light conditions for each exposure of a single leaf to 11CO2 gas. In order to estimate the rate constants of the photosynthesis parameters, the time-activity curves of the input 11CO2 gas and the leaf response were fitted to an appropriate compartmental tracer kinetic model that considers photoassimilation and sucrose export rate constants as influx and efflux, respectively. The data obtained by this method show a reasonable response with respect to the photoenvironment of the leaf, and they are important for discussing photosynthesis with regard to plant physiology and agriculture

Effect of Nitrate on Nodulation and Nitrogen Fixation of Soybean

1.1 Biological nitrogen fixation and nitrogen nutrition in soybean plants Biological nitrogen fixation is one of the most important processes for ecosystem to access available N for all living organisms. Although N2 consists 78% of atmosphere, but the triple bond between two N atoms is very stable, and only a few group of prokaryotes can fix N2 to ammonia by the enzyme nitrogenase. Annual rate of natural nitrogen fixation is estimated about 232 x 106 t, and the 97% depends on biological nitrogen fixation (Bloom, 2011). This exceeds the rate of chemical nitrogen fertilizer uses about 100 x 106 t฀in 2009. Soybean can use N2, though symbiosis with nitrogen fixing soil bacteria, rhizobia, to make root nodules for harboring them. Soybean (Glycine max [L.] Merr.) is a major grain legume crop for feeding humans and livestock. It serves as an important oil and protein source for large population residing in Asia and the American continents. The current global soybean production was 231 x 106 t in 2008 (FAOSTAT). It is a crop predominantly cultivated in U.S.A., Brazil, Argentina and China, which together contribute nearly 87 percent of the total world produce in 2008. Soybean has become the raw materials for diversity of agricultural and industrial uses. Soybean seeds contain a high proportion of protein, about 40% based on dry weight, therefore, they require a large amount of nitrogen to get a high yield. About 8 kg N is required for 100 kg of soybean seed production. Soybean can use atmospheric dinitrogen (N2) by nitrogen fixation of root nodules associated with soil bacteria, rhizobia. Soybean plants can absorb combined nitrogen such as nitrate for their nutrition either from soil mineralized N or fertilizer N. It is well known that heavy supply of nitrogen fertilizer often causes the inhibition of nodulation and nitrogen fixation. Therefore, only a little or no nitrogen fertilizer is

From laboratory to field: OsNRAMP5-knockdown rice is a promising candidate for Cd phytoremediation in paddy fields.

Previously, we reported that OsNRAMP5 functions as a manganese, iron, and cadmium (Cd) transporter. The shoot Cd content in OsNRAMP5 RNAi plants was higher than that in wild-type (WT) plants, whereas the total Cd content (roots plus shoots) was lower. For efficient Cd phytoremediation, we produced OsNRAMP5 RNAi plants using the natural high Cd-accumulating cultivar Anjana Dhan (A5i). Using a positron-emitting tracer imaging system, we assessed the time-course of Cd absorption and accumulation in A5i plants. Enhanced 107Cd translocation from the roots to the shoots was observed in A5i plants. To evaluate the phytoremediation capability of A5i plants, we performed a field experiment in a Cd-contaminated paddy field. The biomass of the A5i plants was unchanged by the suppression of OsNRAMP5 expression; the A5i plants accumulated twice as much Cd in their shoots as WT plants. Thus, A5i plants could be used for rapid Cd extraction and the efficient phytoremediation of Cd from paddy fields, leading to safer food production.

Real-time imaging of nitrogen fixation in an intact soybean plant with nodules using 13N-labeled nitrogen gas

Abstract Real-time images of nitrogen fixation in an intact nodule of hydroponically cultured soybean (Glycine max [L] Merr.) were obtained. In the present study, we developed a rapid method to produce and purify 13N-labeled radioactive nitrogen gas (half life: 9.97 min). 13N was produced from a 16O (p, α) 13N nuclear reaction. The target chamber was filled with CO2 and irradiated for 10 min with protons at an energy of 18.3 MeV and an electric current of 5 μA, which was delivered from a cyclotron. All CO2 in the collected gas was absorbed and removed with powdered soda-lime in a syringe and replaced with helium gas. The resulting gas was injected into gas chromatography and separated and a 35 mL fraction, including the peak of [13N]-nitrogen gas, was collected by monitoring the chromatogram. The obtained gas was mixed with 10 mL of O2 and 5 mL of N2 and used in the tracer experiment. The tracer gas was fed into the underground part of intact nodulated soybean plants and serial images of the distribution of 13N were obtained non-invasively using a positron-emitting tracer imaging system (PETIS). The rates of nitrogen fixation of the six test plants were estimated to be 0.17 ± 0.10 μmol N2 h−1 from the PETIS image data. The decreasing rates of assimilated nitrogen were also estimated to be 0.012 ± 0.011 μmol N2 h−1. In conclusion, we successfully observed nitrogen fixation in soybean plants with nodules non-invasively and quantitatively using [13N]N2 and PETIS.

Kinetic Analysis of Zinc/Cadmium Reciprocal Competitions Suggests a Possible Zn-Insensitive Pathway for Root-to-Shoot Cadmium Translocation in Rice.

BackgroundAmong cereals, rice has a genetic propensity to accumulate high levels of cadmium (Cd) in grains. Xylem-mediated root-to-shoot translocation rather than root uptake has been suggested as the main physiological factor accounting for the genotypic variation observed in Cd accumulation in shoots and grains. Several evidence indicate OsHMA2 – a putative zinc (Zn) transporter – as the main candidate protein that could be involved in mediating Cd- and Zn-xylem loading in rice. However, the specific interactions between Zn and Cd in rice often appear anomalous if compared to those observed in other staple crops, suggesting that root-to-shoot Cd translocation process could be more complex than previously thought. In this study we performed a complete set of competition experiments with Zn and Cd in order to analyze their possible interactions and reciprocal effects at the root-to-shoot translocation level.ResultsThe competition analysis revealed the lack of a full reciprocity when considering the effect of Cd on Zn accumulation, and vice versa, since the accumulation of Zn in the shoots was progressively inhibited by Cd increases, whereas that of Cd was only partially impaired by Zn. Such behaviors were probably dependent on Cd-xylem loading mechanisms, as suggested by: i) the analysis of Zn and Cd content in the xylem sap performed in relation to the concentration of the two metals in the mobile fractions of the roots; ii) the analysis of the systemic movement of 107Cd in short term experiments performed using a positron-emitting tracer imaging system (PETIS).ConclusionsOur results suggest that at least two pathways may mediate root-to-shoot Cd translocation in rice. The former could involve OsHMA2 as Zn2+/Cd2+ xylem loader, whereas the latter appears to involve a Zn-insensitive system that still needs to be identified.

A kinetic analysis of cadmium accumulation in a Cd hyper-accumulator fern, Athyrium yokoscense and tobacco plants

Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron-emitting tracer imaging system under two medium conditions (basal and no-nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no-nutrient condition down-regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt.

Imaging of Carbon Translocation to Fruit Using Carbon-11-Labeled Carbon Dioxide and Positron Emission Tomography

Carbon kinetics into the fruit is an agricultural issue on the growth and development of the sink organs to be harvested. Particularly, photoassimilate translocation and distribution are important topics for understanding the mechanism. In the present work, carbon-11 (11C) labeled photoassimilate translocation into fruits of tomato has been imaged using carbon-11-labeled carbon dioxide and the positron emission tomography (PET). Dynamice PET data of gradual increasing of 11C activity and its distribution is acquired quantitatively in intact plant body. This indicates that the 3-D photoassimilate translocation into the fruits is imaged successfully and carbon kinetics is analyzable to understand the plant physiology and nutrition.

A New Method for Parametric Imaging of Photosynthesis with C-11 Carbon Dioxide and Positron Emitting Tracer Imaging System (PETIS)

The positron emitting tracer imaging system (PETIS) and carbon-11-labeled carbon dioxide (11CO2) can image carbon movement during photosynthesis in a plant leaf, and 11C kinetics make it possible to estimate physiological function parameters in those photosynthesis processes. With an exposure 11CO2 gas to a leaf, PETIS experiments were performed iteratively under dark and light environments on a single leaf. In order to estimate the rate constants of photosynthetic parameters, time activity curves of 11CO2 gas input and leaf response were fitted to an appropriate compartmental tracer kinetic model, which applies influx and efflux for photo-assimilation and sucrose export rate constants respectively. Results obtained from the kinetic analysis are consistent with physiological knowledge and important to discuss photosynthesis in plant physiology and agriculture. In addition, the proposed method in this paper produce parametric images of photosynthetic functions on a pixel-by-pixel basis successfully, in other words, molecular imaging for plant study is demonstrated.

Quantitative analysis of the initial transport of fixed nitrogen in nodulated soybean plants using 15N as a tracer

Abstract The quantitative analysis of the initial transport of fixed isotope 15-nitrogen (15N) in intact nodulated soybean plants (Glycine max [L.] Merr. cv. Williams) was investigated at the vegetative stage (36 days after planting, DAP) and pod-filling stage (91 DAP) by the 15N pulse-chase experiment. The nodulated roots were exposed to N2 gas labeled with a stable isotope 15N for 1 h, followed by 0, 1, 3 and 7 h of exposure with normal air. Plant roots and shoots were separated into three sections (basal, middle and distal parts) with the same length of the main stem or primary root. Approximately 80 and 92% of fixed N was distributed in the basal part of the nodulated roots at the vegetative and pod-filling stages by the end of 1 h of 15N2 exposure, respectively. In addition, about 90% of fixed 15N was retained in the nodules and 10% was exported to root and shoot after 1 h of 15N2 exposure at 91 DAP. The percentage distribution of 15N in the nodules at the pod-filling stage decreased from 90% to 7% during the 7 h of the chase period, and increased in the roots (14%), stems (54%), leaves (12%), pods (10%) and seeds (4%). The 15N distribution was negligible in the distal root segment, suggesting that N fixation activity was negligible and recycling fixed N from the shoot to the roots was very low in the initially short time of the experiment.