Mugineic Acid Research Articles

NUTRITIONAL IMPLICATIONS OF ORGANIC MANAGEMENT IN FRUIT TREE PRODUCTION

The most distinctive feature of tree mineral nutrition in organic farming systems is related to the concept that the target of fertilization is the soil rather than the tree. The best way to achieve this goal is to increase soil organic matter (OM) content through the use of: 1) raw or stabilized manures (i.e. compost obtained from the controlled biological decomposition of organic material), 2) floor permanent grass management, 3) mulches, and 4) abscised leaves, and pruned wood, etc. Fresh OM, such as raw manure (animal and green), cover crop, mulch, etc. with a C:N ratio 20 is incorporated into the soil, microbes use available soil N to break down organic residues and a soil N depletion is expected as well as an increase in soil humus content. Attention must be paid when fresh OM is incorporated into the soil because it may create anoxia conditions. When nutrient deficiencies appear, fertilizers with a fast release of minerals such as blood meal (N = 5-12%), fish meal (N = 5%), natural guano (N = 16%), poultry manure (N = 3.7%) should be used. Stabilized manure and municipal solid waste (MSW) compost present a slower N release rate, but allow a complete 'nutrient cycling' (the breakdown of organic substances, release of energy and matter captured by life processes and their use to stimulate the new growth). In addition, by incorporating MSW composts into the soil there is a sequestration of C that otherwise would follow disposal processes with a potential release of CO 2 in the atmosphere. Iron organic management of fruit trees is a major issue in calcareous soils, where prevention of leaf chlorosis might be achieved by appropriate agronomic techniques that include: introduction of tolerant rootstocks, increase of soil OM content, association with graminaceous such as Festuca spp. known to produce phytosiderophores (i.e. mugineic acid), that are natural Fe chelators, use of blood meal that contains the Fe chelator heme group.

Toward mechanistic elucidation of iron acquisition in barley: efficient synthesis of mugineic acids and their transport activities

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of iron-chelating compounds, mugineic acids (MAs), and by a specific uptake system for MAs-iron(III) complexes. We identified a transporter, HvYS1 (Hordeum vulgare L. yellow stripe 1), that is highly specific for MAs-iron(III) in barley roots. In this article we outline the characterization of HvYS1, and our recent work on the practical syntheses of MAs and investigations into the molecular basis of the specific transport of their iron(III) complexes by HvYS1. 2'-Deoxymugineic acid (DMA) was synthesized in a good overall yield from commercially available Boc-l-allylglycine using a minimal number of short simple operations with minimal protecting groups and work-up/purification procedures. The same strategy was also successfully applied to beta-hydroxy-l-allylglycine, which was obtained by an allylic oxidation of l-allylglycine derivatives, to give MA and 2'-epi-MA efficiently. HvYS1 transported the iron(III) complexes of all three synthetic specimens with efficiency similar to that of a natural mugineic acid complex. With sufficient quantities of MAs in hand, we analyzed the function of HvYS1 and revealed by preparing chimeric transporters that the sixth outer membrane loop of the transporter plays a vital role in substrate specificity.

Complexation of metals by phytosiderophores revealed by CE‐ESI‐MS and CE‐ICP‐MS

CE-ESI-MS and CE-ICP-MS were implemented for studying three phytosiderophores (mugineic acid, epi-mugineic acid and deoxymugineic acid) and their metal complexes. Free ligands and ferric complexes were analyzed using the first methodology, while six free metals (Co(II), Cu(II), Fe(III), Mn(II), Ni(II) and Zn(II)) together with the corresponding complexes were investigated by the latter technique. CE separation was realized at a voltage of +25 kV employing a BGE containing 20 mM ammonium bicarbonate at pH 7.2. Both techniques revealed limits of detection in the high nM to low microM range. Standard additions to hydroponic samples of H. distichon, cv. Bodega (spring barley) showed regression coefficients for the metal-ligand complexes ranging from 0.984 to 0.999. Additionally, results of a competitivity study allowed the determination of relative metal-phytosiderophore complex stability constants of deoxymugineic/mugineic acid.

生物活性天然有機化合物の機能解明を指向した実践的合成手法の開発

The limited supplies of biologically active natural products from natural source have been a severe bottleneck for their application to medical care or mechanistic study of their functions. Synthetic organic chemistry may become a best possible solution of this supply problem by the development of practical synthetic methods suited to the intended molecules. For right-half of halichondrin B, we established a new catalytic cycle of Cr-mediated coupling reaction, new asymmetric Ni/Cr-mediated coupling reaction, surprisingly efficient catalytic Cr-mediated coupling reaction, catalytic Ni/Cr-mediated macrocyclization without use of high-dilution techniques, and effective device for forming polycyclic ring system of halichondrin B. For mugineic acid, we established the practical synthesis of 2'-deoxy mugineic acid with minimal requirements for workup and purification procedure, efficient synthesis of mugineic acid, and synthesis of mugineic acid derivatives as molecular probes. For E-ring of palau'amine, we established Hg(OTf)2-catalyzed olefin cyclization reactions, and its application to the construction of tetra-substituted carbon center possessing nitrogen that corresponds to C16 position of palau'amine.

Recent insights into iron homeostasis and their application in graminaceous crops

Higher plants utilize various mechanisms to maintain iron homeostasis. To acquire sparingly soluble iron from the rhizosphere, graminaceous plants synthesize natural iron (III) chelators known as mugineic acid family phytosiderophores (MAs). Recent research has uncovered various genes involved in iron uptake and translocation, as well as factors regulating the expression of these genes, especially in rice. Manipulation of these molecular components is used to produce transgenic crops with enhanced tolerance to iron deficiency, or with a high seed iron content. Since iron homeostasis is closely linked to that of other mineral elements, an understanding of this phenomenon will serve as the basis for the production of crops with low concentrations of toxic metals and transgenic plants for phytoremediation.

Rice OsYSL15 Is an Iron-regulated Iron(III)-Deoxymugineic Acid Transporter Expressed in the Roots and Is Essential for Iron Uptake in Early Growth of the Seedlings

Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-beta-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth.

Generation and Field Trials of Transgenic Rice Tolerant to Iron Deficiency

Abstract Iron deficiency is a major cause of reduced crop yields worldwide, particularly in calcareous soils. Unlike barley, rice is highly susceptible to iron deficiency because of a low capacity to secrete mugineic acid family phytosiderophores (MAs), which are iron chelators secreted by graminaceous plants. We present an approach toward the generation along with field trials of transgenic rice lines exhibiting increased tolerance to iron deficiency. Cloning barley genes that encode biosynthetic enzymes for MAs enabled us to produce transgenic rice plants by introducing barley MAs biosynthesis-related genes. We tested three transgenic lines possessing barley genomic fragments responsible for MAs biosynthesis in a paddy field experiment on calcareous soil, which revealed tolerance of these lines to low iron availability. We also applied new approaches to generate iron-deficiency-tolerant rice lines, including the introduction of an engineered ferric-chelate reductase gene and manipulation of transcription factor genes regulating the iron deficiency response.

Increase in Iron and Zinc Concentrations in Rice Grains Via the Introduction of Barley Genes Involved in Phytosiderophore Synthesis

Increasing the iron (Fe) and zinc (Zn) concentrations of staple foods, such as rice, could solve Fe and Zn deficiencies, which are two of the most serious nutritional problems affecting humans. Mugineic acid family phytosiderophores (MAs) play a very important role in the uptake of Fe from the soil and Fe transport within the plant in graminaceous plants. To explore the possibility of MAs increasing the Fe concentration in grains, we cultivated three transgenic rice lines possessing barley genome fragments containing genes for MAs synthesis (i.e., HvNAS1, HvNAS1, and HvNAAT-A and HvNAAT-B or IDS3) in a paddy field with Andosol soils. Polished rice seeds with IDS3 inserts had up to 1.40 and 1.35 times higher Fe and Zn concentrations, respectively, compared to non-transgenic rice seeds. Enhanced MAs production due to the introduced barley genes is suggested to be effective for increasing Fe and Zn concentrations in rice grains.

Effect of supplied phytosiderophore on 59Fe absorption and translocation in Fe-deficient barley grown hydroponically in low phosphorus media

Hydroponically grown barley plants (Hordeum vulgare L. cv. Minorimugi) under iron-deficient (–Fe) and high phosphorus (P) conditions (500 µmol L−1) showed Fe chlorosis and lower growth compared with plants grown in –Fe and low P conditions (50, 5 and 0.5 µmol L−1). To understand the physiological role of P in regulating the growth of plants in –Fe medium, we carried out an Fe feeding experiment using four P levels (500, 50, 5 and 0.5 µmol L−1) and phytosiderophores (PS), mugineic acid. Our results suggest that plants grown in a high P medium had higher absorption activity of 59Fe compared with plants grown in low P media, irrespective of the presence or absence of added PS. Translocation of 59Fe from roots to shoots was not affected by the P level. The relative translocation rate of 59Fe increased with decreasing levels of P in the medium. In general, the addition of PS enhanced the absorption of 59Fe and its translocation. Taken together these results suggest that the lower relative translocation rate of Fe in high P plants may be induced by the physiological inactivation of Fe in the roots, and the higher absorption activity of Fe in high P conditions possibly results from the response of barley plants to Fe deficiency.

A Novel NAC Transcription Factor, IDEF2, That Recognizes the Iron Deficiency-responsive Element 2 Regulates the Genes Involved in Iron Homeostasis in Plants

Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.

Investigation of ascorbate-mediated iron release from ferric phytosiderophores in the presence of nicotianamine

Phytosiderophores (PS) are strong iron chelators, produced by graminaceous plants under iron deficiency. The ability of released PS to chelate iron(III), and subsequent uptake of this chelate into roots by YS1-type transport proteins, are well-known. The mechanism of iron release from the stable chelate inside the plant cell, however, is unclear. One possibility involves the reduction of ferric PS in the presence of an iron(II) chelator via ternary complex formation. Here, the conversion of ferric PS species by ascorbate in the presence of the intracellular ligand nicotianamine (NA) has been investigated at cytosolic pH (pH 7.3), leading to the formation of a ferrous NA chelate. This reaction takes place when supplying Fe(III) as a chelate with 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), with the reaction rate decreasing in this order. The progress of the conversion of ferric DMA to ferrous NA was monitored in real-time by high resolution mass spectrometry (FTICR-MS), and the results are complemented by electrochemical measurements (cyclic voltammetry), which allows detecting reactive intermediates and their change with time at high sensitivity. Hence, the combined results of electrochemistry and mass spectrometry indicate an ascorbate-mediated mechanism for the iron release from ferric PS, which highlights the role of ascorbate as a simple, but effective plant reductant.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-008-9292-x) contains supplementary material, which is available to authorized users.

Transgenic rice lines that include barley genes have increased tolerance to low iron availability in a calcareous paddy soil

Iron (Fe) deficiency stress is a widespread problem in agriculture and must be overcome to increase crop yields, particularly in calcareous soils. Unlike barley, rice, one of the three major crops in the world, is very susceptible to low Fe availability because of a low capacity to secrete mugineic acid family phytosiderophores (MAs), which are Fe chelators secreted by graminaceous plants. We tested three transgenic rice lines possessing three barley genes involved in MAs synthesis in a field experiment on a calcareous soil under paddy conditions. Two rice lines, one with a barley gene encoding nicotianamine synthase (NAS) and the other with a barley gene encoding a dioxygenase, referred to as Fe-deficiency specific clone no. 3 (IDS3), showed higher tolerance to low Fe availability under these conditions. The rice line with the IDS3 gene also had increased concentrations of Fe and zinc (Zn) in the grains. These results show that introducing barley genes involved in the synthesis of MAs into rice is an effective and practical method to improve agricultural productivity in calcareous soils.

Specific transporter for iron(III): Phytosiderophore complex involved in iron uptake by barley roots

Abstract Iron (Fe) is an essential element for plant growth. Gramineous plants have generally developed a distinct strategy to efficiently acquire insoluble Fe, which is characterized by the synthesis and secretion of an Fe-chelating substance, phytosiderophore (PS) such as mugineic acid (MA), and by a specific uptake system for Fe(III)-PS complexes. In a previous study, we identified a gene specifically encoding an Fe(III)-PS transporter (HvYS1) in barley. This gene as well as the encoded protein is specifically expressed in the epidermal cells of the roots, and gene expression is greatly enhanced under Fe-deficient conditions. The localization and substrate specificity of HvYS1 indicate that it is a specific transporter in barley roots. In contrast, ZmYS1, which has been reported as an Fe-PS transporter from maize, possesses broad substrate specificity despite a high homology with HvYS1. By assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, we revealed that the outer membrane loop between the 6th and 7th transmembrane regions is essential for the substrate specificity. We also achieved an efficient short-step synthesis of MA and 2'-deoxymugineic acid (DMA). Our new synthetic method enabled us to use them in a large quantity for biological studies.

Identification and localisation of the rice nicotianamine aminotransferase gene OsNAAT1 expression suggests the site of phytosiderophore synthesis in rice

Rice plants (Oryza sativa L.) take up iron using iron-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the biosynthetic pathway of MAs, nicotianamine aminotransferase (NAAT) catalyses the key step from nicotianamine to the 3''-keto form. In the present study, we identified six rice NAAT genes (OsNAAT1-6) by screening a cDNA library made from Fe-deficient rice roots and by searching databases. Among the NAAT homologues, OsNAAT1 belongs to a subgroup containing barley functional NAAT (HvNAAT-A and HvNAAT-B) as well as a maize homologue cloned by cDNA library screening (ZmNAAT1). Northern blot and RT-PCR analysis showed that OsNAAT1, but not OsNAAT2-6, was strongly up-regulated by Fe deficiency, both in roots and shoots. The OsNAAT1 protein had NAAT enzyme activity in vitro, confirming that the OsNAAT1 gene encodes functional NAAT. Promoter-GUS analysis revealed that OsNAAT1 was expressed in companion and pericycle cells adjacent to the protoxylem of Fe-sufficient roots. In addition, expression was induced in all cells of Fe-deficient roots, with particularly strong GUS activity evident in the companion and pericycle cells. OsNAAT1 expression was also observed in the companion cells of Fe-sufficient shoots, and was clearly induced in all the cells of Fe-deficient leaves. These expression patterns highly resemble those of OsNAS1, OsNAS2 and OsDMAS1, the genes responsible for MAs biosynthesis for Fe acquisition. These findings strongly suggest that rice synthesizes MAs in whole Fe-deficient roots to acquire Fe from the rhizosphere, and also in phloem cells to maintain metal homeostasis facilitated by MAs-mediated long-distance transport.

CE of phytosiderophores and related metal species in plants

Phytosiderophores (PS) and the closely related substance nicotianamine (NA) are key substances in metal uptake into graminaceous plants. Here, the CE separation of these substances and related metal species is demonstrated. In particular, the three PS 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), and NA, are separated using MES/Tris buffer at pH 7.3. Moreover, three Fe(III) species of the different PS are separated without any stability problems, which are often present in chromatographic analyses. Also divalent metal species of Cu, Ni, and Zn with the ligands DMA and NA are separated with the same method. By using a special, zwitterionic CE capillary, even the separation of two isomeric Fe(III) chelates with the ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) is possible (i.e., meso-Fe(III)-EDDHA and rac-Fe(III)-EDDHA), and for fast separations of NA and respective divalent and trivalent metal species, a polymer CE microchip with suppressed EOF is described. The proposed CE method is applicable to real plant samples, and enables to detect changes of metal species (Cu-DMA, Ni-NA), which are directly correlated to biological processes.

Contribution of iron associated with high-molecular-weight substances to the maintenance of the SPAD value of young leaves of barley under iron-deficient conditions

In higher plants, it is well known that the retranslocation of iron from old leaves to young leaves is difficult; as a result, iron deficiency leads to interveinal chlorosis, particularly in the young leaves. However, in the case of barley, young chlorotic leaves can grow under conditions of long-term iron deficiency. Previously, we have reported that the distribution and retranslocation characteristics of iron in barley may be better adapted to iron deficiency than those in rice. Furthermore, barley maintained a relatively high chlorophyll index (SPAD value) even when its iron content was not higher than that of rice. In this study, we aimed to predict the chemical form of iron that contributes to the physiologically available iron in barley leaves. To examine the correlation between plant growth and the SPAD value with the amount of fractionated iron, we cultured plant materials in a culture solution containing various iron concentrations. We compared these correlations among barley, rice and sorghum and among three barley cultivars. To compensate for the amount of mugineic acid phytosiderophores (MAs) in the culture solution, we cultured different plant species in the same container. The results revealed that the amount of soluble iron associated with the high-molecular-weight substances (MW >10,000) correlated with the SPAD value of the young barley leaves and the R2 value (determination coefficient) of barley was higher than the values of rice and sorghum.

A Practical Synthesis of the Phytosiderophore 2′‐Deoxymugineic Acid: A Key to the Mechanistic Study of Iron Acquisition by Graminaceous Plants

Ironing out plant uptake: The phytosiderophores mugineic acid (MA) and deoxymugineic acid (DMA) were synthesized in a few steps with minimum use of protecting groups and workup/purification procedures (see scheme; Boc: tert-butoxycarbonyl) and their potencies in transporter-mediated iron(III) acquisition were tested. The sufficient supply of these compounds will enable study of the molecular mechanism of iron acquisition and utilization by graminaceous plants.

The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe‐deficient conditions

Iron (Fe) deficiency is a major abiotic stress in crop production. Although responses to Fe deficiency in graminaceous plants, such as increased production and secretion of mugineic acid family phytosiderophores (MAs), have been described, the gene regulation mechanisms related to these responses are largely unknown. To elucidate the regulation mechanisms of the genes related to Fe acquisition in graminaceous plants, we characterized the Fe-deficiency-inducible basic helix-loop-helix transcription factor OsIRO2 in rice. In yeast cells, OsIRO2 functioned as a transcriptional activator. In rice, overexpression of OsIRO2 resulted in increased MAs secretion, whereas repression of OsIRO2 resulted in lower MAs secretion and hypersensitivity to Fe deficiency. Northern blots revealed that the expression of the genes involved in the Fe(III)-MAs transport system was dependent on OsIRO2. The expression of the genes for nicotianamine synthase, a key enzyme in MAs synthesis, was notably affected by the level of OsIRO2 expression. Microarray analysis demonstrated that OsIRO2 regulates 59 Fe-deficiency-induced genes in roots. Some of these genes, including two transcription factors upregulated by Fe deficiency, possessed the OsIRO2 binding sequence in their upstream regions. OsIRO2 possesses a homologous sequence of the Fe-deficiency-responsive cis-acting elements (IDEs) in its upstream region. We propose a novel gene regulation network for Fe-deficiency responses, including OsIRO2, IDEs and the two transcription factors.

Cadmium uptake in barley affected by iron concentration of the medium: Role of phytosiderophores

Several experiments were conducted using barley (Hordeum vulgare L. cv. Minorimugi) grown hydroponically in a medium containing cadmium (Cd) to clarify the role of phytosiderophores (PS) in chelator-assisted phytoextraction of Cd and the influence of iron (Fe) on Cd uptake. In the first experiment, plants were grown for 7 days in media containing 5 µmol L−1 Cd sulfate (CdSO4), in which the concentration of Fe was 0 or 10 µmol L−1. A marked increase in Cd uptake was obtained in plants grown under the Fe-deficient condition compared with those grown under the Fe-sufficient (10 µmol L−1) condition. In the second experiment, plants were grown for 7 days in media containing 5 µmol L−1 CdSO4, in which the concentration of Fe was 10 or 100 µmol L−1. As a result, the uptake of Cd in plants grown with excess Fe (100 µmol L−1) was significantly lower than the uptake in plants grown under a normal concentration (10 µmol L−1) of Fe. These results indicated that Cd uptake was affected by Fe concentration in the medium and, in particular, a lower concentration of Fe in the media allowed the influx of Cd into plants. Thus, Fe application to the rhizosphere might be effective in reducing Cd concentration in plants. In the third experiment, the ability of solubilizing PS for insoluble Cd mixed with gelatinous Fe was examined. The results showed that PS could solubilize Cd from sulfide of Cd (CdS) gel. In the fourth experiment, roots of Fe-deficient or Fe-sufficient plants were fed with 0 or 10 µmol L−1 mugineic acid (MA), one of the PS, for 4 h in media containing 5 µmol L−1 CdSO4. The uptake of Cd by the plants was not increased with MA. In the fifth experiment, the amount of PS release in Fe-deficient barley suffering from Cd toxicity was measured. Release of PS was partially reduced by 0.05 µmol L−1 CdSO4 and largely reduced by 0.5 or 5 µmol L−1 CdSO4. Based on these results, it was suggested that, when PS were released by roots of grasses to the rhizosphere, PS could mobilize insoluble Cd around the roots. It was also suggested that PS could not convey Cd into the root cells in the form of a PS–Cd complex. Thus, the main role of PS for chelator-assisted phytoextraction of Cd might be the collection of Cd to the rhizosphere, resulting in the enhancement of Cd concentration in the rhizosphere.