Cadmium accumulation dynamics in the rice endosperm during grain filling revealed by autoradiography.

Cadmium (Cd) is one of the environmental pollutants contaminated in our food. Several previous reports showed that rice polishing cannot be efficient to reduce Cd content in white rice, implying the characteristic Cd distribution in rice grain. However, Cd distribution has not been fully elucidated so far. Herein, 109Cd radiotracer experiment was performed using the rice seedlings at various time points after flowering to obtain autoradiographs of the brown rice to visually understand the Cd transport and distribution during the grain-filling process. It was shown that 109Cd accumulated in the outermost area of the brown rice, and also in the middle part of the starchy endosperm, resulting in the appearance of the double circle distribution pattern, which was not observed in the autoradiographs of 65Zn. The inner circle of 109Cd located around the center of the endosperm was developed particularly at around 8 and 10 days after flowering. After this period, 109Cd started to deposit at the outer part of the endosperm, which was also found in the autoradiograph of 14C-sucrose. Considering the physiology of grain development, the contribution of water transport and protein synthesis in the endosperm on the characteristic Cd distribution pattern was hypothesized.

Mutations in RZF1, a zinc-finger protein, reduce magnesium uptake in roots and translocation to shoots in rice.

Magnesium (Mg) homeostasis is critical for maintaining many biological processes, but little information is available to comprehend the molecular mechanisms regulating Mg concentration in rice (Oryza sativa). To make up for the lack of information, we aimed to identify mutants defective in Mg homeostasis through a forward genetic approach. As a result of the screening of 2,825 M2 seedlings mutated by ion-beam irradiation, we found a rice mutant that showed reduced Mg content in leaves and slightly increased Mg content in roots. Radiotracer 28Mg experiments showed that this mutant, named low-magnesium content 1 (LMGC1), has decreased Mg2+ influx in the root and Mg2+ translocation from root to shoot. Consequently, LMGC1 is sensitive to the low Mg condition and prone to develop chlorosis in the young mature leaf. The MutMap method identified a 7.4-kbp deletion in the LMGC1 genome leading to a loss of two genes. Genome editing using CRISPR-Cas9 further revealed that one of the two lost genes, a gene belonging to the RanBP2-type zinc-finger family that we named RanBP2-TYPE ZINC FINGER1 (OsRZF1), was the causal gene of the low Mg phenotype. OsRZF1 is a nuclear protein and may have a fundamental role in maintaining Mg homeostasis in rice plants.

Mutagenesis analysis of GMN motif in Arabidopsis thaliana Mg2+ transporter MRS2-1.

Magnesium is an important nutrient for plants, but much is still unknown about plant Mg2+ transporters. Combining with the structural prediction of AlphaFold2, we used mutagenesis and 28Mg uptake assay to study the highly conserved "GMN" motif of Arabidopsis thaliana MRS2-1 (AtMRS2-1) transporter. We demonstrated that the glycine and methionine in GMN motif are essential for AtMRS2-1 to transport Mg2+.

Visualization of phosphorus re-translocation and phosphate transporter expression profiles in a shortened annual cycle system of poplar.

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P are considered an important strategy to support the vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using real-time radioisotope imaging and the macro- and micro-autoradiography. We analysed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under 'a shortened annual cycle system', which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of 32 P and/or 33 P shifted from young leaves and the apex to the lower stem and roots. The radioisotope P re-translocated from a leaf was stored in phloem and xylem parenchyma cells and redistributed to new shoots after dormancy. Seasonal expression profile of phosphate transporters (PHT1, PHT5 and PHO1 family) was obtained in the same system. Our results reveal the seasonal P re-translocation routes at the organ and tissue levels and provide a foothold for elucidating its molecular mechanisms.

Short-Term Magnesium Deficiency Triggers Nutrient Retranslocation in Arabidopsis thaliana.

Magnesium (Mg) is essential for many biological processes in plant cells, and its deficiency causes yield reduction in crop systems. Low Mg status reportedly affects photosynthesis, sucrose partitioning and biomass allocation. However, earlier physiological responses to Mg deficiency are scarcely described. Here, we report that Mg deficiency in Arabidopsis thaliana first modified the mineral profile in mature leaves within 1 or 2 days, then affected sucrose partitioning after 4 days, and net photosynthesis and biomass production after 6 days. The short-term Mg deficiency reduced the contents of phosphorus (P), potassium, manganese, zinc and molybdenum in mature but not in expanding (young) leaves. While P content decreased in mature leaves, P transport from roots to mature leaves was not affected, indicating that Mg deficiency triggered retranslocation of the mineral nutrients from mature leaves. A global transcriptome analysis revealed that Mg deficiency triggered the expression of genes involved in defence response in young leaves.

What you can see by developing real-time radioisotope imaging system for plants: from water to element and CO2 gas imaging.

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.

Visualization of 14CO2 gas fixation by plants.

Using the real-time radioisotope imaging system (RRIS), we present the carbon dioxide gas fixation process of a soybean plant applying the 14C-labeled gas. When 14CO2 gas was supplied to the selected mature leaf, the fixed carbon, photosynthate, was transferred and accumulated to the younger leaves preferentially within 24 h. When 14CO2 gas was supplied to the younger leaves, fixed carbon was hardly moved. In the case of the pods, fixed 14CO2 gas in the leaf was preferentially transferred to the closest pod.

Magnesium uptake characteristics in Arabidopsis revealed by 28Mg tracer studies.

MAIN CONCLUSION: The Mg2+ uptake system in Arabidopsis roots is Gd3+- and Fe2+-sensitive, and responds to a changing Mg2+ concentration within 1 h with the participation of AtMRS2 transporters. Magnesium (Mg2+) absorption and the mechanism regulating its activity have not been clarified yet. To address these issues, it is necessary to reveal the characteristics of Mg2+ uptake in roots. Therefore, we first investigated the Mg2+ uptake characteristics in roots of 1-week-old Arabidopsis plants using 28Mg. The Mg2+ uptake system in roots was up-regulated within 1 h in response to the low Mg2+ condition. This induction was inhibited in Arabidopsis "mitochondrial RNA splicing 2/magnesium transport" mutants atmrs2-4/atmgt6 and atmrs2-7/atmgt7, while the expression of AtMRS2-4/AtMGT6 and AtMRS2-7/AtMGT7 genes in the Arabidopsis wild-type was not responsive to Mg2+ conditions. In addition, the Mg deficiency-induced Mg2+ uptake system was shut-down within 5 min when Mg2+ was resupplied to the environment. An inhibition study showed that the constitutive mechanism functioning in Mg2+ uptake under Mg2+ sufficient conditions was sensitive to a number of divalent and trivalent cations, particularly Gd3+ and Fe2+, but not to K+.

Agricultural aspects of radiocontamination induced by the Fukushima nuclear accident - A survey of studies by the Univ. of Tokyo Agricultural Dept. (2011-2016).

Immediately after the Fukushima nuclear power plant accident, a team of 40-50 researchers at the Graduate School of Agricultural and Life Sciences at the University of Tokyo began to analyze the behavior of radioactive materials in the fallout regions. The fallout has remained in situ and become strongly adsorbed within the soil over time. 137Cs was found to bind strongly to the fine clay, weathered biotite, and organic matter in the soil; therefore, it has not mobilized from mountainous regions, even after heavy rainfall. In farmland, the quantity of 137Cs in the soil absorbed by crop plants was small. The downward migration of 137Cs in soil is now estimated at 1-2 mm/year. The intake of 137Cs by trees occurred through the bark and not from the roots. This report summarizes the findings of research across a wide variety of agricultural specialties.

Research with radiation and radioisotopes to better understand plant physiology and agricultural consequences of radioactive contamination from the Fukushima Daiichi nuclear accident.

Research carried out by me and my group over the last almost four decades are summarized here. The main emphasis of my work was and continues to be on plant physiology using radiation and radioisotopes. Plants live on water and inorganic elements. In the case of water, we developed neutron imaging methods and produced 15O-labeled water (half-life 2 min) and applied them to understand water circulation pattern in the plant. In the case of elements, we developed neutron activation analysis methods to analyze a large number of plant tissues to follow element specific distribution. Then, we developed real-time imaging system using conventional radioisotopes for the macroscopic and microscopic observation of element movement. After the accident in Fukushima Daiichi nuclear power plant, we, the academic staff of Graduate School, have been studying agricultural effects of radioactive fallout; the main results are summarized in two books published by Springer.

Short day length-induced decrease of cesium uptake without altering potassium uptake manner in poplar.

Short day length-induced alteration of potassium (K) localization in perennial trees is believed to be a mechanism for surviving and adapting to severe winters. To investigate the relationship between cesium (Cs) and K localizations, a model tree poplar, hybrid aspen T89, was employed. Under short day length conditions, the amount of 137Cs absorbed through the root and translocated to the root was drastically reduced, but 42K was not. Potassium uptake from the rhizosphere is mediated mainly by KUP/HAK/KT and CNGC transporters. In poplar, however, these genes were constantly expressed under short-day conditions except for a slight increase in the expression a KUP/HAK/KT gene six weeks after the onset of the short-day treatment. These results indicated that the suppression of 137Cs uptake was triggered by short day length but not regulated by competitive Cs+ and K+ transport. We hypothesize that there are separately regulated Cs+ and K+ transport systems in poplar.

Agricultural implications of the Fukushima nuclear accident.

More than 4 years has passed since the accident at the Fukushima Nuclear Power Plant. Immediately after the accident, 40 to 50 academic staff of the Graduate School of Agricultural and Life Sciences at the University of Tokyo created an independent team to monitor the behavior of the radioactive materials in the field and their effects on agricultural farm lands, forests, rivers, animals, etc. When the radioactive nuclides from the nuclear power plant fell, they were instantly adsorbed at the site where they first touched; consequently, the fallout was found as scattered spots on the surface of anything that was exposed to the air at the time of the accident. The adsorption has become stronger over time, so the radioactive nuclides are now difficult to remove. The findings of our study regarding the wide range of effects on agricultural fields are summarized in this report.

A novel role for the root cap in phosphate uptake and homeostasis.

The root cap has a fundamental role in sensing environmental cues as well as regulating root growth via altered meristem activity. Despite this well-established role in the control of developmental processes in roots, the root cap's function in nutrition remains obscure. Here, we uncover its role in phosphate nutrition by targeted cellular inactivation or phosphate transport complementation in Arabidopsis, using a transactivation strategy with an innovative high-resolution real-time (33)P imaging technique. Remarkably, the diminutive size of the root cap cells at the root-to-soil exchange surface accounts for a significant amount of the total seedling phosphate uptake (approximately 20%). This level of Pi absorption is sufficient for shoot biomass production (up to a 180% gain in soil), as well as repression of Pi starvation-induced genes. These results extend our understanding of this important tissue from its previously described roles in environmental perception to novel functions in mineral nutrition and homeostasis control.

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,(22)Na,(28)Mg,(32)P-phosphate,(35)S-sulfate,(42)K,(45)Ca,(54)Mn and(137)Cs, was employed as an ion tracer, ion movement from root to shoot over 24 h was clearly observed. The movements of(22)Na,(42)K,(32)P,(35)S and(137)Cs were fast so that they spread to the tip of stems. In contrast, high accumulation of(28)Mg,(45)Ca and(54)Mn 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,(14)CO2 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,(14)C 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.

Performance and Limitations of Phosphate Quantification: Guidelines for Plant Biologists.

Phosphate (Pi) is a macronutrient that is essential for plant life. Several regulatory components involved in Pi homeostasis have been identified, revealing a very high complexity at the cellular and subcellular levels. Determining the Pi content in plants is crucial to understanding this regulation, and short real-time(33)Pi uptake imaging experiments have shown Pi movement to be highly dynamic. Furthermore, gene modulation by Pi is finely controlled by localization of this ion at the tissue as well as the cellular and subcellular levels. Deciphering these regulations requires access to and quantification of the Pi pool in the various plant compartments. This review presents the different techniques available to measure, visualize and trace Pi in plants, with a discussion of the future prospects.

Cesium adsorption/desorption behavior of clay minerals considering actual contamination conditions in Fukushima.

Cesium adsorption/desorption experiments for various clay minerals, considering actual contamination conditions in Fukushima, were conducted using the (137)Cs radioisotope and an autoradiography using imaging plates (IPs). A 50 µl solution containing 0.185 ~ 1.85 Bq of (137)Cs (10(-11) ~ 10(-9 )molL(-1) of (137)Cs) was dropped onto a substrate where various mineral particles were arranged. It was found that partially-vermiculitized biotite, which is termed "weathered biotite" (WB) in this study, from Fukushima sorbed (137)Cs far more than the other clay minerals (fresh biotite, illite, smectite, kaolinite, halloysite, allophane, imogolite) on the same substrate. When WB was absent on the substrate, the amount of (137)Cs sorbed to the other clay minerals was considerably increased, implying that selective sorption to WB caused depletion of radiocesium in the solution and less sorption to the coexisting minerals. Cs-sorption to WB continued for about one day, whereas that to ferruginous smectite was completed within one hour. The sorbed (137)Cs in WB was hardly leached with hydrochloric acid at pH 1, particularly in samples with a longer sorption time. The presence/absence of WB sorbing radiocesium is a key factor affecting the dynamics and fate of radiocesium in Fukushima.

A chemical genetic strategy identify the PHOSTIN, a synthetic molecule that triggers phosphate starvation responses in Arabidopsis thaliana.

Plants display numerous strategies to cope with phosphate (Pi)-deficiency. Despite multiple genetic studies, the molecular mechanisms of low-Pi-signalling remain unknown. To validate the interest of chemical genetics to investigate this pathway we discovered and analysed the effects of PHOSTIN (PSN), a drug mimicking Pi-starvation in Arabidopsis. We assessed the effects of PSN and structural analogues on the induction of Pi-deficiency responses in mutants and wild-type and followed their accumulation in plants organs by high pressure liquid chromotography (HPLC) or mass-spectrophotometry. We show that PSN is cleaved in the growth medium, releasing its active motif (PSN11), which accumulates in plants roots. Despite the overaccumulation of Pi in the roots of treated plants, PSN11 elicits both local and systemic Pi-starvation effects. Nevertheless, albeit that the transcriptional activation of low-Pi genes by PSN11 is lost in the phr1;phl1 double mutant, neither PHO1 nor PHO2 are required for PSN11 effects. The range of local and systemic responses to Pi-starvation elicited, and their dependence on the PHR1/PHL1 function suggests that PSN11 affects an important and early step of Pi-starvation signalling. Its independence from PHO1 and PHO2 suggest the existence of unknown pathway(s), showing the usefulness of PSN and chemical genetics to bring new elements to this field.

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 root's 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.

Inspections of radiocesium concentration levels in rice from Fukushima Prefecture after the Fukushima Dai-ichi Nuclear Power Plant accident.

We summarize the inspections of radiocesium concentration levels in rice produced in Fukushima Prefecture, Japan, for 3 years from the nuclear accident in 2011. In 2011, three types of verifications, preliminary survey, main inspection, and emergency survey, revealed that rice with radiocesium concentration levels over 500 Bq/kg (the provisional regulation level until March 2012 in Japan) was identified in the areas north and west of the Fukushima nuclear power plant. The internal exposure of an average adult eating rice grown in the area north of the nuclear plant was estimated as 0.05 mSv/year. In 2012, Fukushima Prefecture authorities decided to investigate the radiocesium concentration levels in all rice using custom-made belt conveyor testers. Notably, rice with radiocesium concentration levels over 100 Bq/kg (the new standard since April 2012 in Japan) were detected in only 71 and 28 bags out of the total 10,338,000 in 2012 and 11,001,000 in 2013, respectively. We considered that there were almost no rice exceeding 100 Bq/kg produced in Fukushima Prefecture after 3 years from the nuclear accident, and the safety of Fukushima's rice were ensured because of the investigation of all rice.

Monitoring free-living Japanese Bush Warblers (Cettia diphone) in a most highly radiocontaminated area of Fukushima Prefecture, Japan.

The Fukushima-Daiichi Nuclear Power Plant (F1NPP) accident is an IAEA level 7 event, the same as that of Chernobyl, while the amount of radionuclides released is not comparable. Radioactivity attributed to the F1NPP accident was detected 250 km away from the F1NPP. Although we have not yet systematically studied the effect of radionuclides on the environment and wildlife, one of three Japanese Bush Warblers (Cettia diphone), captured in Akaugi district in August 2011, was observed to have a conspicuous lesion near the cloaca, which is rare in Japan. All of the birds' feathers were strongly contaminated. Further study is needed to determine the significance of this result. We emphasize the importance of continuing assessment of the effects of the F1NPP accident on wildlife.