ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals

Among higher plants graminaceous species have the unique ability to efficiently acquire iron from alkaline soils with low iron solubility by secreting phytosiderophores, which are hexadentate metal chelators with high affinity for Fe(III). Iron(III)-phytosiderophores are subsequently taken up by roots via YS1 transporters, that belong to the OPT oligopeptide transporter family. Despite its physiological importance at alkaline pH, uptake of Fe-phytosiderophores into roots of wild-type maize plants was greater at acidic pH and sensitive to the proton uncoupler CCCP. To access the mechanism of Fe-phytosiderophore acquisition, ZmYS1 was expressed in an iron uptake-defective yeast mutant and in Xenopus oocytes, where ZmYS1-dependent Fe-phytosiderophore transport was stimulated at acidic pH and sensitive to CCCP. Electrophysiological analysis in oocytes demonstrated that Fephytosiderophore transport depends on proton cotransport and on the membrane potential, which allows ZmYS1-mediated transport even at alkaline pH. We further investigated substrate specificity and observed that ZmYS1 complemented the growth defect of the zinc uptake-defective yeast mutant zap1 and transported various phytosiderophore-bound metals into oocytes, including zinc, copper, nickel, and, at a lower rate, also manganese and cadmium. Unexpectedly, ZmYS1 also transported Ni(II), Fe(II), and Fe(III) complexes with nicotianamine, a structural analog of phytosiderophores, which has been shown to act as an intracellular metal chelator in all higher plants. Our results show that ZmYS1 encodes a proton-coupled broad-range metal-phytosiderophore transporter that additionally transports Fe- and Ni-nicotianamine. These biochemical properties indicate a novel role of YS1 transporters for heavy metal homeostasis in plants.     

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.     

The effect of pH,electrolytes and temperature on the rhizosphere geochemistry of phytosiderophores

Background and aims

Graminaceous plants are grown worldwide as staple crops under a variety of climatic and soil conditions. They release phytosiderophores for Fe acquisition (Strategy II). Aim of the present study was to uncover how the rhizosphere pH, background electrolyte and temperature affect the mobilization of Fe and other metals from soil by phytosiderophores.

Methods

For this purpose a series of kinetic batch interaction experiments with the phytosiderophore 2′-deoxymugineic acid (DMA), a calcareous clay soil and a mildly acidic sandy soil were performed. The temperature, electrolyte concentration and applied electrolyte cation were varied. The effect of pH was examined by applying two levels of lime and Cu to the acidic soil.

Results

Fe mobilization by DMA increased by lime application, and was negatively affected by Cu amendment. Mobilization of Fe and other metals decreased with increasing ionic strength, and was lower for divalent than for monovalent electrolyte cations at equal ionic strength, due to higher adsorption of metal-DMA complexes to the soil. Metal mobilization rates increased with increasing temperature leading to a faster onset of competition; Fe was mobilized faster, but also became depleted faster at higher temperature. Temperature also affected biodegradation rates of metal-DMA complexes.

Conclusion

Rhizosphere pH, electrolyte type and concentration and temperature can have a pronounced effect on Strategy II Fe acquisition by affecting the time and concentration ‘window of Fe uptake’ in which plants can benefit from phytosiderophore-mediated Fe uptake.

     

Response of barley plants to Fe deficiency and Cd contamination as affected by S starvation

Both Fe deficiency and Cd exposure induce rapid changes in the S nutritional requirement of plants. The aim of this work was to characterize the strategies adopted by plants to cope with both Fe deficiency (release of phytosiderophores) and Cd contamination [production of glutathione (GSH) and phytochelatins] when grown under conditions of limited S supply. Experiments were performed in hydroponics, using barley plants grown under S sufficiency (1.2 mM sulphate) and S deficiency (0 mM sulphate), with or without Fe(III)-EDTA at 0.08 mM for 11 d and subsequently exposed to 0.05 mM Cd for 24 h or 72 h. In S-sufficient plants, Fe deficiency enhanced both root and shoot Cd concentrations and increased GSH and phytochelatin levels. In S-deficient plants, Fe starvation caused a slight increase in Cd concentration, but this change was accompanied neither by an increase in GSH nor by an accumulation of phytochelatins. Release of phytosiderophores, only detectable in Fe-deficient plants, was strongly decreased by S deficiency and further reduced after Cd treatment. In roots Cd exposure increased the expression of the high affinity sulphate transporter gene (HvST1) regardless of the S supply, and the expression of the Fe deficiency-responsive genes, HvYS1 and HvIDS2, irrespective of Fe supply. In conclusion, adequate S availability is necessary to cope with Fe deficiency and Cd toxicity in barley plants. Moreover, it appears that in Fe-deficient plants grown in the presence of Cd with limited S supply, sulphur may be preferentially employed in the pathway for biosynthesis of phytosiderophores, rather than for phytochelatin production.     

Uptake kinetics of iron-phytosiderophores in two maize genotypes differing in iron efficiency

Iron inefficiency in the maize ( Zea mays L.) mutant ysl is caused by a defect in the uptake system for Fe-phytosiderophores. To characterize this defect further, the uptake kinetics of Fe-phytosiderophores in ysl was compared to the Fe-efficient maize cultivar Alice. Short-term uptake of ⁵⁹Fe-labeled Fe-deoxymugineic acid (Fe-DMA) was measured over a concentration range of 0.03 to 300 μM. Iron uptake in Fe-deficient plants followed Michaelis-Menten kinetics up to about 30 μM and was linear at higher concentrations, indicating two kinetically distinct components in the uptake of Fe-phytosiderophores. The saturable component had similar K_m (∼ 10 μM) in both genotypes. In contrast. V_max was 5.5 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in Alice, but only 0.6 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in _ysl. Uptake experiments with double-labeled ⁵⁹Fe-[¹⁴C]DMA suggest that in both cultivars Fe-DMA was taken up by the roots as the intact chelate. The results indicate the existence of a high-affinity and a low-affinity uptake system mediating Fe-phytosiderophore transport across the root plasma membrane in maize. Apparently, the mutation responsible for Fe inefficiency in ysl affected high-affected uptake and led to a decrease in activity and/or number of Fe-phytosiderophore transporters.     

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.     

Effect of Al on the phytosiderophore-mediated solubilization of Fe and uptake of Fe-phytosiderophore complex in wheat (Triticum aestivum)

The effect of Aluminum (Al) on phytosiderophore-mediated solubilization of insoluble Fe and the uptake of phytosiderophore-Fe³⁺ complex was examined in wheat ( Triticum aestivum L. cv. Atlas 66). Al addition did not affect the Fe solubilization by 2'-deoxymugineic acid (DMA), although Cu addition significantly inhibited the solubilization capacity. Addition of ten times more Al than Fe to the solution of DMA-Fe³⁺ complex did not decrease the absorption of the DMA-Fe³⁺ complex at 375 nm. Furthermore, NMR study indicated that Al did not shift the proton chemical shifts of DMA. All these results suggest that Al could not form a complex with the phytosiderophore, and is thereby unlikely to affect the process of phytosiderophore-mediated solubilization of Fe. Exposure of root to Al up to 100 μ M for 3 h did not inhibit the DMA-Fe³⁺ uptake by the roots, but longer pretreatment (>6 h) inhibited the uptake of the DMA-Fe³⁺ by more than 50%. Neither the uptake of DMA-Fe³⁺ nor root elongation was inhibited by 24 h pretreatment with 10 μ M Al, but both uptake and root elongation were inhibited by higher Al (>20 μ M ) pretreatment. These results suggest that Al did not directly block the transport of the phytosiderophore-Fe³⁺ complex, and that the decreased uptake of the phytosiderophore-Fe³⁺ complex resulted from the roots being damaged by Al.     

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.     

A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis

Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.     

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.     

Identification of two novel phytosiderophores secreted by perennial grasses

It has been suggested that some perennial grasses secrete phytosiderophores in response to iron (Fe) deficiency, but the compounds have not been identified. Here, we identified and characterized the phytosiderophores secreted by two perennial grasses, Lolium perenne cv. Tove and Poa pratensis cv. Baron. Root exudates were collected from the roots of Fe-deficient grasses and then purified with various chromatographies. The structure of the purified compounds was determined using both nuclear magnetic resonance and fast atom bombardment mass spectrometry. Both species secreted phytosiderophores in response to Fe deficiency, and the amount of phytosiderophores secreted increased with the development of Fe deficiency. The type of phytosiderophores secreted differed with plant species; L. perenne cv. Tove secreted 3-epihydroxy-2'-deoxymugineic acid (epiHDMA), 2'-deoxymugineic acid (DMA) and an unknown compound, whereas P. pratensis cv. Baron secreted DMA, avenic acid A (AVA) and an unknown compound. Purification and subsequent analysis with nuclear magnetic resonance and mass led to identification of the two novel phytosiderophores; 3-hydroxy-2'-deoxymugineic acid (HDMA) from L. perenne, and 2'-hydroxyavenic acid A (HAVA) from P. pratensis. Both novel phytosiderophores have similar chelating activity to known phytosiderophores.     

A sensitive LC-ESI-Q-TOF-MS method reveals novel phytosiderophores and phytosiderophore-iron complexes in barley

? The direct analysis of phytosiderophores (PSs) and their metal complexes in plants is critical to understanding the biological functions of different PSs. Here we report on a rapid and highly sensitive liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) method for the direct and simultaneous determination of free PSs and their ferric complexes in plants. ? In addition to previously reported PSs - deoxymugineic acid (DMA), mugineic acid (MA) and epihydroxymugineic acid (epi-HMA) - two more PSs, avenic acid (AVA) and hydroxyavenic acid (HAVA), were identified by this method in roots of Hordeum vulgare cv Himalaya and in root exudates under iron (Fe) deficiency. ? The two identified PSs could be responsible for Fe acquisition under Fe deficiency because of their relative abundance and ability to form ferric complexes in secreted root exudates. ? This LC-ESI-Q-TOF-MS method greatly facilitates the identification of free PSs and PS-Fe complexes in one plant sample.     

Structural element responsible for the Fe(III)-phytosiderophore specific transport by HvYS1 transporter in barley

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter for Fe(III)-phytosiderophores, involved in primary iron acquisition from soils in barley roots. In contrast, Zea mays yellow stripe 1 (ZmYS1) in maize possesses broad substrate specificity, despite a high homology with HvYS1. Here we revealed, by assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, that the outer membrane loop between the sixth and seventh transmembrane regions is essential for substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an alpha-helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.     

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 material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Motofumi Suzuki and Takashi Tsukamoto equally contributed to this work.     

Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters

Background and Aims

Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency.

Methods

The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography.

Key Results

Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated.

Conclusions

PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis.     

Evidence for a specific uptake system for iron phytosiderophores in roots of grasses

Roots of grasses in response to iron deficiency markedly increase the release of chelating substances (`phytosiderophores') which are highly effective in solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds by formation of Fe<sup>III</sup>phytosiderophores. In barley (Hordeum vulgare L.), the rate of iron uptake from Fe<sup>III</sup>phytosiderophores is 100 to 1000 times faster than the rate from synthetic Fe chelates (e.g. Fe ethylenediaminetetraacetate) or microbial Fe siderophores (e.g. ferrichrome). Reduction of Fe<sup>III</sup> is not involved in the preferential iron uptake from Fe<sup>III</sup>phytosiderophores by barley. This is indicated by experiments with varied pH, addition of bicarbonate or of a strong chelator for Fe<sup>II</sup> (e.g. batho-phenanthrolinedisulfonate). The results indicate the existence of a specific uptake system for Fe<sup>III</sup>phytosiderophores in roots of barley and all other graminaceous species. In contrast to grasses, cucumber plants (Cucumis sativus L.) take up iron from Fe<sup>III</sup>phytosiderophores at rates similar to those from synthetic Fe chelates. Furthermore, under Fe deficiency in cucumber, increased rates of uptake of Fe<sup>III</sup>phytosiderophores are based on the same mechanism as for synthetic Fe chelates, namely enhanced Fe<sup>III</sup> reduction and chelate splitting. Two strategies are evident from the experiments for the acquisition of iron by plants under iron deficiency. Strategy I (in most nongraminaceous species) is characterized by an inducible plasma membrane-bound reductase and enhancement of H<sup>+</sup> release. Strategy II (in grasses) is characterized by enhanced release of phytosiderophores and by a highly specific uptake system for Fe<sup>III</sup>phytosiderophores. Strategy II seems to have several ecological advantages over Strategy I such as solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds in the rhizosphere, and less inhibition by high pH. The principal differences in the two strategies have to be taken into account in screening methods for resistance to `lime chlorosis'.