A specific transporter for iron(III)-phytosiderophore in barley roots

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron-chelating phytosiderophore, mugineic acid (MA), and by a specific uptake system for iron(III)-phytosiderophore complexes. We identified a gene specifically encoding an iron-phytosiderophore transporter (HvYS1) in barley, which is the most tolerant species to iron deficiency among graminaceous plants. HvYS1 was predicted to encode a polypeptide of 678 amino acids and to have 72.7% identity with ZmYS1, a first protein identified as an iron(III)-phytosiderophore transporter in maize. Real-time RT-PCR analysis showed that the HvYS1 gene was mainly expressed in the roots, and its expression was enhanced under iron deficiency. In situ hybridization analysis of iron-deficient barley roots revealed that the mRNA of HvYS1 was localized in epidermal root cells. Furthermore, immunohistological staining with anti-HvYS1 polyclonal antibody showed the same localization as the mRNA. HvYS1 functionally complemented yeast strains defective in iron uptake on media containing iron(III)-MA, but not iron-nicotianamine (NA). Expression of HvYS1 in Xenopus oocytes showed strict specificity for both metals and ligands: HvYS1 transports only iron(III) chelated with phytosiderophore. The localization and substrate specificity of HvYS1 is different from those of ZmYS1, indicating that HvYS1 is a specific transporter for iron(III)-phytosiderophore involved in primary iron acquisition from soil in barley roots.     

Phytosiderophore release from nodal,primary, and complete root systems in maize

Some maize (Zea mays L.) hybrids grown in high pH soil in Nebraska suffer from severely reduced yields caused by iron (Fe) deficiency chlorosis. Hybrids which recover from early season Fe-deficiency chlorosis and yield well are termed Fe-efficient or tolerant. Most Fe-efficient gramineous species respond to Fe-deficiency stress by releasing phytosiderophores (mugineic acid and its derivatives) into the rhizosphere, thereby increasing Fe availability and uptake of the Fe³⁺-phytosiderophore complex via a high affinity uptake system. Field-grown Fe-efficient maize recovers from Fe-deficiency chlorosis at a stage when nodal roots have become the dominant root system. Quantifying phytosiderophore release from hydroponically grown plants has been proposed as a viable alternative to time-consuming and variable field trials and has been used successfully to delineate among Fe-efficient and Fe-inefficient lines of oat (Avena sativa L.) and wheat (Triticum aestivum L.). Our objectives were (1) to determine if phytosiderophore release differed between nodal- and primary-root systems of maize, and (2) to compare phytosiderophore release from 12 hybrids. Root exudates secreted during daily 4-h collections were analyzed for their Fe-solubilizing ability, which was equated to phytosiderophore release. Nodal root systems released significantly more phytosiderophore than primary- or complete-root systems. In early experiments, an Fe-efficient hybrid (P3279) released more phytosiderophore from nodal roots than an Fe-inefficient hybrid (P3489). Tests of an additional 10 hybrids showed that phytosiderophore release varied significantly among the cultivars but did not clearly distinguish between hybrids classified as Fe-efficient or Fe-inefficient in individual company trials. We recommend using nodal roots when studying Fe-stress response mechanisms in maize.     

Phytosiderophores revisited: 2′-deoxymugineic acid-mediated iron uptake triggers nitrogen assimilation in rice (Oryza sativa L.) seedlings

Poaceae plants release phytosiderophores into the rhizosphere in order to chelate iron (Fe), which often exists in insoluble forms especially under high pH conditions. The impact of phytosiderophore treatment at the physiological and molecular levels in vivo remains largely elusive, although the biosynthesis of phytosiderophores and the transport of phytosiderophore-metal complexes have been well studied. We recently showed that the application of 30 μM of the chemically synthesized phytosiderophore 2′-deoxymugineic acid (DMA) was sufficient for apparent full recovery of otherwise considerably reduced growth of hydroponic rice seedlings at high pH. Moreover, unexpected induction of high-affinity nitrate transporter gene expression as well as nitrate reductase activity indicates that the nitrate response is linked to Fe homeostasis. These data shed light on the biological relevance of DMA not simply as a Fe chelator, but also as a trigger that promotes plant growth by reinforcing nitrate assimilation.     

The binding of aluminum to mugineic acid and related compounds as studied by potentiometric titration

The phytosiderophores, mugineic acid (MA) and epi-hydroxymugineic acid (HMA), together with a related compound, nicotianamine (NA), were investigated for their ability to bind Al(III). Potentiometric titration analysis demonstrated that MA and HMA bind Al(III), in contrast to NA which does not under normal physiological conditions. With MA and HMA, in addition to the Al complex (AlL), the protonated (AlLH) and deprotonated (AlLH₋₁) complexes were identified from an analysis of titration curves, where L denotes the phytosiderophore form in which all the carboxylate functions are ionized. The equilibrium formation constants of the Al(III) phytosiderophore complexes are much smaller than those of the corresponding Fe(III) complexes. The higher selectivity of phytosiderophores for Fe(III) over Al(III) facilitates Fe(III) acquisition in alkaline conditions where free Al(III) levels are higher than free Fe(III) levels.     

Goethite Dissolution in the Presence of Phytosiderophores: Rates, Mechanisms, and the Synergistic Effect of Oxalate

The purpose of this study was the elucidation of the chemical mechanism of an important process in iron acquisition by graminaceous plants: the dissolution of iron oxides in the presence of phytosiderophores. We were particularly interested in the effects of diurnal root exudation of phytosiderophores and of the presence of other organic ligands in the rhizosphere of graminaceous plants on the dissolution mechanism. Phytosiderophores of the type 2′-deoxymugineic acid (DMA) were purified from the root exudates of wheat plants (Triticum aestivum L. cv. Tamaro). DMA-promoted dissolution of goethite under steady-state and non-steady-state conditions and its dependence on pH, adsorbed DMA concentration, and the presence of the organic ligand oxalate were studied. We show that dissolution of goethite by phytosiderophores follows a surface controlled ligand promoted dissolution mechanism. We also found that oxalate, an organic ligand commonly found in rhizosphere soils, has a synergistic effect on the steady-state dissolution of goethite by DMA. Under non-steady-state addition of the phytosiderophore, mimicking the diurnal exudation pattern of phytosiderophore release, a fast dissolution of iron is triggered in the presence of oxalate. To investigate the efficiency of these mechanisms in plant iron acquisition, wheat plants were grown on a substrate amended with goethite as only iron source. The chlorophyll status of these plants was similar to iron-fertilized plants and significantly higher than in plants grown in iron free nutrient solutions. This demonstrates that wheat can efficiently mobilize iron, even from well crystalline goethite that is usually considered unavailable for plant nutrition.     

Transgenic Petunia with the Iron(III)-Phytosiderophore Transporter Gene Acquires Tolerance to Iron Deficiency in Alkaline Environments

Iron is an essential nutrient for all plants. However, terrestrial plants often suffer from iron deficiency in alkaline soil due to its extremely low solubility. Alkaline soil accounts for about 30% of all cultivated ground in the world. Plants have evolved two distinct strategies, I and II, for iron uptake from the soil. Dicots and non-graminaceous monocots use Strategy I, which is primarily based on the reduction of iron(III) to iron(II) and the uptake of iron(II) by the iron-regulated transporter, IRT1. In contrast, graminaceous plants use Strategy II to efficiently acquire insoluble iron(III). Strategy II comprises the synthesis and secretion of iron-chelating phytosiderophores, such as mugineic acids and the Yellow Stripe 1 transporter proteins of the iron(III)-phytosiderophore complex. Barley, which exhibits the highest tolerance to iron deficiency in alkaline soil among graminaceous plants, utilizes mugineic acids and the specific iron(III)-mugineic acids transporter, HvYS1. In this study, we established the transgenic plant Petunia hybrida, which originally had only Strategy I, by introducing the HvYS1 transporter gene derived from barley. When the transgenic plants were grown hydroponically in media containing the iron(III)-2′-deoxymugineic acid complex, free 2′-deoxymugineic acid and its iron(III) complex were detected in the root extract of the transgenic plant by electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry. The growth of the transgenic petunia was significantly better than that of the control host in alkaline conditions. Consequently, the transgenic plant acquired a significantly enhanced tolerance to alkaline hydroponic media in the presence of the iron(III)-2′-deoxymugineic acid complex. Furthermore, the flower color of the transgenic plant deepened. The results showed that iron-phytosiderophore complexes and their transporters can potentially be utilized to overcome the worldwide iron uptake problems to diverse plant species that are found in areas with alkaline conditions.     

Is the release of phytosiderophores in zinc-deficient wheat plants a response to impaired iron utilization?

The effect of zinc nutritional status on the time course of phytosiderophore release, and uptake of iron and translocation of iron to the shoot, was studied in nutrient solution cultures for two cultivars of wheat ( Triticum aestivum . cv. Aroona: T. durum , cv. Duratit) differing in their susceptibility to zinc deficiency. In the zinc-efficient cultivar Aroona, under zinc deficiency translocation of iron from roots to shoot was significantly decreased in 13- and 15-day-old plants, whereas release of phytosiderophores was enhanced when the plants were 16 days old. As zinc deficiency became more severe in older plains, translocation of iron to the shoot was further decreased and release of phytosiderophores was further enhanced. Resupplying zinc in nutrient solution to zinc-deficient plants significantly increased the translocation of iron to the shoot after 48 and 72 h. Concomitantly the release of phytosiderophores was repressed. The other cultivar Durati classified as zinc-inefficient in field observations differed from cv. Aroona by showing a lower rate of phytosiderophore release under Zinc deficiency, and a less impaired translocation of iron to the shoot. Foliar application of iron citrate to zinc-deficient Aroona plants repressed the release of phytosiderophores and increased iron concentrations in shoot and roots. Application of ⁵⁵Fe to the leaves demonstrated that retranslocation of iron from the shoot to the roots was not affected by the zinc nutritional status. It is concluded that enhanced release of phytosiderophores in zinc-deficient wheat plants was induced primarily by impaired trans-location of iron lo the shoot.     

Effect of salt on physiological responses of barley to iron deficiency.

Iron chlorosis is very common on alkaline soils such as calcareous ones, since iron availability is limited by high pH. Under these conditions of iron deficiency, graminaceous plant species induce special mechanisms for iron acquisition, involving enhanced release of iron chelators called phytosiderophores. On the other hand, it is known that most of salt soils have alkaline pH. So, plants growing on this kind of soils are often subjected simultaneously to salinity and iron deficiency. This work aimed at (i) studying the physiological responses of barley (Hordeum vulgare L.) to iron deficiency, and (ii) evaluating the effect of salt on the iron nutrition and the phytosiderophore release. For this purpose, seedlings of Hordeum vulgare L. were cultivated under controlled conditions, either in a complete nutrient solution with or without NaCl, or in an iron free nutrient solution containing or not NaCl. The plant morphological aspect, chlorophyll content of young leaves, iron status, biomass production, and phytosiderophore release by roots were assessed. Plants subjected to Fe deficiency exhibited a severe chlorosis, accompanied by a significant biomass reduction. These plants developed more lateral roots than the control with a highly stimulated phytosiderophore release. However, the latter was greatly diminished when iron deficiency was associated to salinity. A depressive effect of salt on iron acquisition in plants subjected only to salt stress which was also observed and further confirmed by the important decrease of efficiency in iron acquisition. These results suggest that salinity may reduce capacity of plants to acquire iron from alkaline soils by inhibiting phytosiderophore release.     

Localization of phytosiderophore release and of iron uptake along intact barley roots

Under iron deficiency the release of so-called phytosiderophores by roots of barley plants ( Hordeum vulgare L. cv. Europa) was greater by a factor of 10 to 50 compared to iron-sufficient plants. This enhanced release occurred particularly in apical zones of the seminal roots and in the lateral root zones. Under iron deficiency, uptake rates for iron, supplied as FeIII phytosiderophore, increased by a factor of ca 5 as compared to iron-sufficient plants. This enhanced uptake rate for iron was also much more pronounced in apical than in basal root zones. In contrast, with supply of the synthetic iron chelate, FelII EDDHA (ferric diaminoethane-N, N-di- o -hydroxyphenyl acetic acid), the Fe deficiency-enhanced uptake rates for iron were only small and similar along the roots, except for the lateral root zones. The high selectivity of barley roots for uptake and translocation of FeIII phytosiderophores compared with FeIII EDDHA is reflected by the fact that, at the same external concentration (2 μ M ), rates of uptake and translocation of iron from FeIII phytosiderophores were between 100 (Fe-sufficient) and 1 000 times higher (Fe-deficient plants) than from FeIII EDDHA. The relatively high rates of uptake and particularly of translocation of iron supplied as FeIII EDDHA in the zone of lateral root formation strongly suggest an apoplastic pathway of radial transport of the synthetic iron chelate into the stele in this root zone.
The results demonstrate that apical root zones are the main sites both for Fe deficiency-enhanced release of phytosiderophores and for uptake and translocation of iron supplied as FeIII phytosiderophores.     

The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice

Iron is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire iron in the soil. Phytosiderophores chelate and solubilize insoluble iron hydroxide in the soil. Subsequently, plants take up iron-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals, including iron. In this study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-β-glucuronidase revealed that TOM2 was expressed in tissues involved in metal translocation, whereas TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, whereas TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared with non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released ¹⁴C-labeled deoxymugineic acid, the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of deoxymugineic acid, which is required for normal plant growth.     

Light-mediated release of phytosiderophores in wheat and barley under iron or zinc deficiency

The effect of varied light intensity (50 – 600 mol m-2 s-1) on the rate of phytosiderophore release was studied under zinc (Zn) deficiency using a bread (Triticum aestivum L. cv. Aroona) and a durum wheat cultivar (Triticum durum Desf. cv. Durati) differing in zinc (Zn) efficiency and under iron (Fe) deficiency using a barley cultivar (Hordeum vulgare L. Europe). Plants were grown under controlled environmental conditions in nutrient solution for 15 days (wheat plants) or 11 days (barley plants). Phytosiderophore release was determined by measuring capacity of root exudates to mobilize copper (Cu) from a Cu-loaded resin.With increasing light intensity visual Zn deficiency symptoms such as whitish-brown lesions on leaf blade developed rapidly and severely in wheat, particularly in the durum cultivar Durati. In wheat plants supplied well with Zn, increases in light intensity from 100 to 600 mol m-2 s-1 did not clearly affect the rate of phytosiderophore release. However, under Zn deficiency increases in light intensity markedly enhanced release of phytosiderophores in Zn-deficient Aroona, but not in Zn-inefficient Durati. When Fe-deficient barley cultivar Europe was grown first at 220 mol m-2 s-1 and then exposed to 600 mol m-2 s-1 for 24 and 48 h, the rate of release of phytosiderophores was enhanced about 4-fold and 7-fold, respectively. Transfer of Fe-deficient plants from 600 to 50 mol m-2 s-1 for 48 h reduced the rate of release of phytosiderophores by a factor of 7. The effect of light on phytosiderophore release was similar regardless of whether the rate of phytosiderophore release was expressed per plant or per unit dry weight of roots.The results demonstrate a particular role of light intensity in phytosiderophore release from roots under both Zn and Fe deficiency. It is suggested that in the studies concerning the role of phytosiderophore release in expression of Zn or Fe efficiency among and within cereals, a special attention should be given to the light conditions.     

The Role of Ligand Exchange in the Uptake of Iron from Microbial Siderophores by Gramineous Plants

The siderophore rhizoferrin, produced by the fungus Rhizopus arrhizus, was previously found to be as an efficient Fe source as Fe-ethylenediamine-di(o-hydroxphenylacetic acid) to strategy I plants. The role of this microbial siderophore in Fe uptake by strategy II plants is the focus of this research. Fe-rhizoferrin was found to be an efficient Fe source for barley (Hordeum vulgare L.) and corn (Zea mays L.). The mechanisms by which these Gramineae utilize Fe from Fe-rhizoferrin and from other chelators were studied. Fe uptake from 59Fe-rhizoferrin, 59Fe-ferrioxamine B, 59Fe-ethylenediaminetetraacetic acid, and 59Fe-2[prime]-deoxymugineic acid by barley plants grown in nutrient solution at pH 6.0 was examined during periods of high (morning) and low (evening) phytosiderophore release. Uptake and translocation rates from Fe chelates paralleled the diurnal rhythm of phytosiderophore release. In corn, however, similar uptake and translocation rates were observed both in the morning and in the evening. A constant rate of the phytosiderophore's release during 14 h of light was found in the corn cv Alice. The results presented support the hypothesis that Fe from Fe-rhizoferrin is taken up by strategy II plants via an indirect mechanism that involves ligand exchange between the ferrated microbial siderophore and phytosiderophores, which are then taken up by the plant. This hypothesis was verified by in vitro ligand-exchange experiments.     

Molecular evidence for phytosiderophore‐induced improvement of iron nutrition of peanut intercropped with maize in calcareous soil

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe‐deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1‐like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)–DMA. These results imply that in alkaline soil, Fe(III)–DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.     

Root-microbial effects on plant iron uptake from siderophores and phytosiderophores

Collaborative experiments were conducted to determine whether microbial populations associated with plant roots may artifactually affect the rates of Fe uptake and translocation from microbial siderophores and phytosiderophores. Results showed nonaxenic maize to have 2 to 34-fold higher Fe-uptake rates than axenically grown plants when supplied with 1 μM Fe as either the microbial siderophore, ferrioxamine B (FOB), or the barley phytosiderophore, epi-hydroxymugineic acid (HMA). In experiments with nonsterile plants, inoculation of maize or oat seedlings with soil microorganisms and amendment of the hydroponic nutrient solutions with sucrose resulted in an 8-fold increase in FOB-mediated Fe-uptake rates by Fe-stressed maize and a 150-fold increase in FOB iron uptake rates by Fe-stressed oat, but had no effect on iron uptake by Fe-sufficient plants. Conversely, Fe-stressed maize and oat plants supplied with HMA showed decreased uptake and translocation in response to microbial inoculation and sucrose amendment. The ability of root-associated microorganisms to affect Fe-uptake rates from siderophores and phytosiderophores, even in short-term uptake experiments, indicates that microorganisms can be an unpredictable confounding factor in experiments examining mechanisms for utilization of microbial siderophores or phytosiderophores under nonsterile conditions.     

Iron acquisition by phytosiderophores contributes to cadmium tolerance

Based on the ability of phytosiderophores to chelate other heavy metals besides iron (Fe), phytosiderophores were suggested to prevent graminaceous plants from cadmium (Cd) toxicity. To assess interactions between Cd and phytosiderophore-mediated Fe acquisition, maize (Zea mays) plants were grown hydroponically under limiting Fe supply. Exposure to Cd decreased uptake rates of 59Fe(III)-phytosiderophores and enhanced the expression of the Fe-phytosiderophore transporter gene ZmYS1 in roots as well as the release of the phytosiderophore 2'-deoxymugineic acid (DMA) from roots under Fe deficiency. However, DMA hardly mobilized Cd from soil or from a Cd-loaded resin in comparison to the synthetic chelators diaminetriaminepentaacetic acid and HEDTA. While nano-electrospray-high resolution mass spectrometry revealed the formation of an intact Cd(II)-DMA complex in aqueous solutions, competition studies with Fe(III) and zinc(II) showed that the formed Cd(II)-DMA complex was weak. Unlike HEDTA, DMA did not protect yeast (Saccharomyces cerevisiae) cells from Cd toxicity but improved yeast growth in the presence of Cd when yeast cells expressed ZmYS1. When supplied with Fe-DMA as a Fe source, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing a cauliflower mosaic virus 35S-ZmYS1 gene construct showed less growth depression than wild-type plants in response to Cd. These results indicate that inhibition of ZmYS1-mediated Fe-DMA transport by Cd is not related to Cd-DMA complex formation and that Cd-induced phytosiderophore release cannot protect maize plants from Cd toxicity. Instead, phytosiderophore-mediated Fe acquisition can improve Fe uptake in the presence of Cd and thereby provides an advantage under Cd stress relative to Fe acquisition via ferrous Fe.     

Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released (14)C-labeled deoxymugineic acid but not (14)C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.     

OsYSL16 plays a role in the allocation of iron

Graminaceous plants acquire iron by secreting mugineic acid family phytosiderophores into the rhizosphere and taking up complexes of iron and phytosiderophores through YSL (yellow stripe 1-like) transporters. Rice OsYSL15 is a transporter of the iron(III)-2'-deoxymugineic acid complex. OsYSL16 has 85?% similarity to both OsYSL15 and the iron(II)-nicotianamine transporter OsYSL2. In the present study, we show that OsYSL16 functionally complemented a yeast mutant defective in iron uptake when grown on medium containing iron(III)-deoxymugineic acid, but not when grown on medium containing iron(II)-nicotianamine. OsYSL16-knockdown seedlings were smaller than wild-type seedlings when only iron(III)chloride was supplied as an iron source. The iron concentration in shoots of OsYSL16-knockdown plants was similar to that of the wild type; however, they showed more severe chlorosis than wild-type plants under iron-deficient conditions. Furthermore, OsYSL16-knockdown plants accumulated more iron in the vascular bundles of the leaves. Expression of the OsYSL16 promoter fused to the β-glucuronidase gene showed that OsYSL16 is expressed in the root epidermis and vascular bundles of whole plants. The expression was typically observed around the xylem. In the vascular bundles of unelongated nodes, it was detected in the xylem of old leaves and the phloem of new leaves. Graminaceous plants translocate iron from the roots to old leaves mainly via the xylem and to new leaves mainly via the phloem. Our results suggest that OsYSL16 plays a role in the allocation of iron(III)-deoxymugineic acid via the vascular bundles.     

Mutation in nicotianamine aminotransferase stimulated the Fe(II) acquisition system and led to iron accumulation in rice

Higher plants acquire iron (Fe) from the rhizosphere through two strategies. Strategy II, employed by graminaceous plants, involves secretion of phytosiderophores (e.g. deoxymugineic acid in rice [Oryza sativa]) by roots to solubilize Fe(III) in soil. In addition to taking up Fe in the form of Fe(III)-phytosiderophore, rice also possesses the strategy I-like system that may absorb Fe(II) directly. Through mutant screening, we isolated a rice mutant that could not grow with Fe(III)-citrate as the sole Fe source, but was able to grow when Fe(II)-EDTA was supplied. Surprisingly, the mutant accumulated more Fe and other divalent metals in roots and shoots than the wild type when both were supplied with EDTA-Fe(II) or grown under water-logged field conditions. Furthermore, the mutant had a significantly higher concentration of Fe in both unpolished and polished grains than the wild type. Using the map-based cloning method, we identified a point mutation in a gene encoding nicotianamine aminotransferase (NAAT1), which was responsible for the mutant phenotype. Because of the loss of function of NAAT1, the mutant failed to produce deoxymugineic acid and could not absorb Fe(III) efficiently. In contrast, nicotianamine, the substrate for NAAT1, accumulated markedly in roots and shoots of the mutant. Microarray analysis showed that the expression of a number of the genes involved in Fe(II) acquisition was greatly stimulated in the naat1 mutant. Our results demonstrate that disruption of deoxymugineic acid biosynthesis can stimulate Fe(II) acquisition and increase iron accumulation in rice.     

Short communication. Biosynthesis of phytosiderophores in several Triticeae species with different genomes

To examine variation in phytosiderophore biosynthesis in Triticeae, phytosiderophores were investigated in wild and cultivated species of wheat and barley with different genomes. All wheats tested including hexaploid (AABBDD), tetraploid (AABB),and diploid (AA or DD) lines produced only one phytosiderophore, 2-deoxymugineic acid. The phytosiderophores biosynthesized in wild barleys varied among species. Using substitution-type triticale lines and wheat-barley addition lines. it was revealed that, in triticale, genes for the biosynthesis of both mugineic and hydroxymugineic acids were located in the long arm of chromosome 5R and that, in barley, the gene for production of mugineic acid was located in the long arm of chromosome 4H.     

Comparative evaluation of iron solubilizing substances (phytosiderophores) released by oats and corn: Iron-efficient and iron-inefficient plants

Release of phytosiderophores from barley (Hordeum vulgare L.) in response to Fe-deficiency stress prompted further testing of other graminaceous (grass) species for phytosiderophore release and results have prompted characterization of these plants into a Strategy II designation. This classification denotes an enhanced release of phytosiderophore in response to Fe-deficiency stress with a concomitant uptake of Fe by the plant. The objective of this study was to determine if Fe-inefficient and Fe-efficient corn (Zea mays L.) differ in their release of Fe solubilizing substances in response to Fe-deficiency stress. We have not identified the specific structure of these substances but refer to them as phytosiderophores to further characterize their behavior. By our indirect method, there was no measurable release of Fe solubilizing substances (phytosiderophores) from either the Fe-efficient WF9 or the Fe-inefficient ys₁ corn despite WF9 being greener and apparently more Fe efficient than ys₁. Fe-efficient Coker 227 oats (Avena byzantina C. Koch.) has been found to release a phytosiderophore whereas the Fe-inefficient TAM 0-312 does not. Iron-stressed Coker 227 oats released Fe solubilizing substances when grown in the same solution with WF9 corn which resulted in greening and Fe uptake by WF9 corn. Iron efficiency in these two graminaceous species appears to be controlled by different mechanisms.