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

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

Analysis of Galacturonic Acid and Oligogalacturonic Acid by High-Performance Liquid Chromatography

ペクチン酸の酵素分解物の迅速分析を目的として、ガラクツロン酸とオリゴガラクツロン酸のHPLCによる分析について検討した。分子サイズとイオン排除を分離機構に持つ強酸性陽イオン交換樹脂のShodex SUGAR SH1821カラムを用い、0.005N硫酸を溶離液としてモノ～テトラガラクツロン酸の分離状態を調べた。カラム温度40℃、流速1.0ml/minで、トリガラクツロン酸とテトラガラクツロン酸間に部分的な重なりはあったが、ほぼ満足な結果を得た。分析所要時間は約9分であった。流速を500μl/minに下げるとさらに良好な分離が得られた。このときの分析所要時間は約18分であった。梗準試料(モノ～テトラガラクツロン酸)の検量線は広い濃度範園で良好な波線性を示した。本法による検出限界は、ガラクツロン酸ではおおよそ0.1μgであり、少なくとも0.5μg以上のガラクツロン酸が定量的に精度よく測定できることがわかった。定量精度を検定するために、ガラクツロン酸0.5～4μgに対する測定値(n=5)について標準偏差と変動係数を求めたが、前者はいずれの場合も1以下であり、後者も1～2%と低い値を示し、精度は非常に良好であることがわかった。Kluyveromyces fragilisのendo-PGによるペクチン酸の分解経過を反応初期から終期に至る段階について追跡調査した。流速1.0ml/minの条件で、モノからヘキサまでの一連のオリゴガラクツロン酸が区別でき、またそれらの数的変化の追跡も容易なことから、本法が、ペクチン酸の酵素分解物の分析や分解経過の追跡に非常に有効な分析手段であることを確認した。本法は、ペクチナーゼの作用様式やペクチン質の構造研究において、今後、ますます有用になるものと思われる。A mixture of mono- ~ tetragalacturonic acid standards was separated by both size and ion exclusion mechanisms on a strong cation-exchange column ( 8 x 300 mm) of Shodex SUGAR SH1821 with 0.005 N sulfuric acid as elueut. Each separation was complete in 9 mill at a flow rate of 1.0 ml/min. The relationship between the peak response and concentration was strictly linear throughout the entire concentration range studied (0.5 μg/sample to 50 μg/sample of individual standards): a differential refractometer was used as detector. At an attenuation of 1 x 10^-6 RIU, the coefficients of variation of the peak height for repeated injections (n = S) were 2.3 and 1.l% for 0.5 and l.0 μg/sample of galacturonic acid, respectively. The detection limit of the uronic acid was estimated to be 0.1μg. The method was applied to follow the course of hydrolysis of pectic acid by an Endopolygalactutronase, and proved to be a very powerful tool for studying the mechanism of action of pectic enzymes. A series of oligogalacturonic acids having chain lengths of two to six units could be distinguish from each other in the digest at an early stage of the hydrolysis (10 mill after the start); the major products were tetra-, penta-, and hexagalacturonic acids. At a final stage (40 hr after the start), only three components of mono-, di-, and trigalacturonic acids were detected in the digest; the major product was trigalacturonic acid, amounting to 63% of the total pectic acid used. A total of 92% of the original pectic acid was recovered in the three components

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 Fe(2+) transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe(3+) reduction activity. A yeast Fe(3+) 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 Fe(3+) 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 Fe(3+) chelate-reductase activity of rice plants that normally have low endogenous levels confers resistance to Fe deficiency

Use of positron emitting tracer system for measuring the effect of salinity on temporal and spatial distribution of 11C tracer and coupling between source and sink organs.

Salinity stress affects photosynthate partitioning between sources and sinks of plants, but how it affects on these systems is less understood. Because sources and sinks are closely knitted, any adverse effect under sub-optimal condition on one part is often misinterpreted for the other. Knowledge on regulation of carbon partitioning is indispensable for stress resistance and good plant growth. In the present study, alteration of carbon partitioning in tomato plants (lycopersicon esculentum L. cv. Momotarou) under saline (NaCl) environment was studied by feeding radioactive 11C and stable 13C isotopes. Pulse chases were conducted for measuring spatial and temporal distributions of 13C. 13C was measured by standard conventional technique, but 11C distribution was monitored using by PETIS. Salt stress resulted in reduced carbon translocation towards roots. Majority of the photosynthate accumulated in the leaf. We have also observed that the reduction in translocation of carbon occurred well before salt stress symptoms of reduced photosynthesis and plant growth in salt exposed plants. The effect on sink activity also showed by decrease in stem diameter and reduced photosynthetic activity. In addition, PETIS analysis of 11C translocation indicates that carbon translocation to roots was inhibited under salt conditions without direct effect of leaf Na accumulation and osmotic stress These results suggest that NaCl has direct effects on plants inhibiting carbon partitioning within few hours of salt solution exposure without inhibition of source activity