Shinpei Matsuhashi

Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil

Iron (Fe) deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability due to high soil pH. Rice plants use a well documented phytosiderophore-based system (Strategy II) to take up Fe from the soil and also possess a direct Fe2+ transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe3+ reduction activity. A yeast Fe3+ chelate-reductase gene refre1/372, selected for better performance at high pH, was fused to the promoter of the Fe-regulated transporter, OsIRT1, and introduced into rice plants. The transgene was expressed in response to a low-Fe nutritional status in roots of transformants. Transgenic rice plants expressing the refre1/372 gene showed higher Fe3+ chelate-reductase activity and a higher Fe-uptake rate than vector controls under Fe-deficient conditions. Consequently, transgenic rice plants exhibited an enhanced tolerance to low-Fe availability and 7.9× the grain yield of nontransformed plants in calcareous soils. This report shows that enhancing the Fe3+ chelate-reductase activity of rice plants that normally have low endogenous levels confers resistance to Fe deficiency.

Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants.

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less 62Zn-DMA than 62Zn2+. Importantly, supply of 62Zn-DMA rather than 62Zn2+ increased the translocation of 62Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil.

52Fe Translocation in Barley as Monitored by a Positron-Emitting Tracer Imaging System (PETIS) : Evidence for the Direct Translocation of Fe from Roots to Young Leaves via Phloem

The real-time translocation of iron (Fe) in barley (Hordeum vulgare L. cv. Ehimehadaka no. 1) was visualized using the positron-emitting tracer 52Fe and a positron-emitting tracer imaging system (PETIS). PETIS allowed us to monitor Fe translocation in barley non-destructively under various conditions. In all cases, 52Fe first accumulated at the basal part of the shoot, suggesting that this region may play an important role in Fe distribution in graminaceous plants. Fe-deficient barley showed greater translocation of 52Fe from roots to shoots than did Fe-sufficient barley, demonstrating that Fe deficiency causes enhanced 52Fe uptake and translocation to shoots. In the dark, translocation of 52Fe to the youngest leaf was equivalent to or higher than that under the light condition, while the translocation of 52Fe to the older leaves was decreased, in both Fe-deficient and Fe-sufficient barley. This suggests the possibility that the mechanism and/or pathway of Fe translocation to the youngest leaf may be different from that to the older leaves. When phloem transport in the leaf was blocked by steam treatment, 52Fe translocation from the roots to older leaves was not affected, while 52Fe translocation to the youngest leaf was reduced, indicating that Fe is translocated to the youngest leaf via phloem in addition to xylem. We propose a novel model in which root-absorbed Fe is translocated from the basal part of the shoots and/or roots to the youngest leaf via phloem in graminaceous plants.

Enhancement of Antimicrobial Activity of Chitosan by Irradiation

Antimicrobial activity of irradiated chitosan was studied against Escherichia coli B/r. Irradiation of chitosan at 100 kGy under dry conditions was effective in increasing the activity, and inhibited the growth of E coli completely. The molecular weight of chitosan significantly decreased with the increase in irradiation dose, whereas the relative surface charge of chitosan was decreased only 3% by 100 kGy irradiation. Antimicrobial activity assay of chitosan fractionated according to molecular weight showed that 1 x 10 5 -3 x 10 5 fraction was most effective in suppressing the growth of E coli. This fraction comprised only 8% of the 100 kGy irradiated chitosan. On the other hand, chitosan whose molecular weight was less than 1 x 10 5 had no activity. The results show that low dose irradiation, specifically 100 kGy, of chitosan gives enough degradation to increase its antimicrobial activity as a result of a change in molecular weight.

Uptake and transport of positron-emitting tracer (18F) in plants☆

Abstract The transport of a positron-emitting isotope introduced into a plant was dynamically followed by a special observation apparatus called ‘Positron-Emitting Tracer Imaging System’ to observe the damage and recovery functions of plants in vivo. In the system, annihilation γ-rays from the positron emitter are detected with two planar detectors (5 × 6 cm2). The water containing ca. 5 MBq/ml of 18F was fed to the cut stem of soybean for 2 min and then the images of tracer activity were recorded for 30–50 min. When the midrib of a leaf near the petiole was cut just before measurement, the activity in the injured leaf was decreased but detected even at the apex. This result suggests that the damaged leaf recovered the uptake of water through the lamina. Maximum tracer activities in leaves of unirradiated plant were observed within 10 min, whereas those of irradiated plant at 100 Gy were observed after over 25 min. The final activity of irradiated plant after 30 min was lower than that of unirradiated plant. In case of beans, there was a difference in the absorption behavior of the 18F-labeled water between unirradiated and irradiated samples. These results show that the system is effective to observe the uptake and transportation of water containing positron emitting tracer for the study of damage and recovery functions of plants.

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

52Mn translocation in barley monitored using a positron‐emitting tracer imaging system

Abstract Until now, the real-time uptake and movement of manganese (Mn), an essential plant nutrient, has not been documented in plants. In this study, the real-time translocation of Mn in barley (Hordeum vulgare L. cv. Ehimehadaka no. 1) was visualized using the positron-emitting tracer 52Mn and a positron-emitting tracer imaging system (PETIS). PETIS allowed the non-destructive monitoring of Mn translocation in barley under various conditions. In all cases, 52Mn first accumulated in the discrimination center (DC) at the basal portion of the shoot, suggesting that this region may play an important role in Mn distribution in graminaceous plants. Manganese-deficient barley showed greater translocation of 52Mn from roots to shoots than did Mn-sufficient barley, demonstrating that Mn deficiency causes enhanced Mn uptake and loading into vascular bundles. In contrast, the translocation of 52Mn from roots to shoots was suppressed in Mn-excess barley. In these plants, the uptake of Mn may be suppressed or Mn may accumulate in the intercellular organelles of root cells, resulting in low rates of Mn translocation to shoots. In Mn-sufficient barley, the dark treatment did not suppress the translocation of 52Mn to the youngest leaf, suggesting that the translocation of Mn to the youngest leaf is independent of the transpiration stream. When 52Mn was supplied to the cut end of an expanded leaf, 52Mn was transported to the DC within 27 min and then retranslocated to roots and other leaves. Our results show that the translocation of Mn from the roots to the DC depends passively on water flow, but actively on the Mn transporter(s).

Quantitative Modeling of Photoassimilate Flow in an Intact Plant Using the Positron Emitting Tracer Imaging System (PETIS)

The photoassimilate flow in an intact plant stem was imaged in real-time and its dynamics was quantitatively described using the Positron Emitting Tracer Imaging System (PETIS). Radioactive 11CO2 was fed to a leaf of an intact broad bean (Vicia faba L.) plant, together with air containing an ambient concentration of non-radioactive carrier CO2 gas. Movies of flow of the 11C-labeled photoassimilates in the plant body were captured with PETIS. Here we demonstrate that the average flow speeds and the distribution ratios of photoassimilates in the respective nodes and internodes of the observed stem can be estimated by the transfer function analysis, one of the mathematical modeling methods. We also estimated the changes in the spatial distribution of the average flow speeds in the same stem when the fed leaf was exposed to enriched carrier CO2 gas.

Visualization of 15O-water flow in tomato and rice in the light and dark using a positron-emitting tracer imaging system (PETIS)

Abstract 15P-water flow from the roots to the top in tomato (Lycopersicon esculentum Mill.) and rice (Oryza sativa L.) plants was visualized with time using a positron-emitting tracer imaging system (PETIS). The 15O-water flow was switched on by light and completely stopped in the dark. The flow rate in the stem of tomato and the shoot of rice at a light intensity of 500 μmol·m−2·s−1 was 1.9 and 0.4 cm min−1, respectively.

Detection and characterization of nitrogen circulation through the sieve tubes and xylem vessels of rice plants

Nitrogen movement through the xylem vessels and sieve tubes in rice plants was studied using xylem and phloem sap analysis in combination with stable and radioactive nitrogen isotope techniques.More than 90% of nitrogen was translocated in the sieve tubes of rice plants as amino acids. When 15N (99.6 atom%) was applied as a nitrate to the root, 15N first appeared in phloem sap of the leaf sheath within 10 minutes and increased to 37 atom% excess 5 hours after the experiment had started. In long-term experiments, 63% of nitrogen in the phloem sap of the leaf sheath and 15% in that of the uppermost internode came from nitrogen absorbed within the last 24 hours and 50 hours, respectively.To obtain information about the more rapid circulation of nitrogen in the plant, radioactive 13N was used as a tracer. A positron-emitting tracer imaging system was used to show that 13N was transferred to the leaf sheath within 8 minutes of its application to the roots. Analysis of the xylem sap of the leaf sheath showed that when the nitrate was applied to the roots, most of the nitrogen in the xylem was transported as a nitrate.These data showed that phloem and xylem sap analysis together with the stable and radioactive nitrogen techniques provide a good method for the detection of nitrogen cycles in plants.