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

Authors

Errata

This article is corrected by:

  1. Errata: Correction Volume 61, Issue 1, 188, Article first published online: 18 December 2009

*(fax +81 75 962 2115; email murata@sunbor.or.jp).

Summary

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.

Introduction

Iron (Fe) is an essential element for all organisms. Because animals ultimately depend on plants for their iron, the primary uptake of iron by plants from soil is very important for all living beings (Curie and Briat, 2003; Grusak and DellaPenna, 1999). However, crops suffer from iron-deficiency stress due to the extremely low solubility of iron in alkaline soils, which cover about one-third of the soils (Mori, 1999).

Plants have evolved two distinct iron uptake strategies by the roots (Marschner et al., 1986). Most plants, including dicots and non-graminaceous monocots, transport ferrous ion [Fe(II)] from soils into root cells via a transporter after reduction from ferric ion [Fe(III)] on the plasma membrane (Eide et al., 1996; Robinson et al., 1999; strategy I). In contrast, some graminaceous plants have a distinct iron uptake system (Römheld and Marschner, 1986; strategy II). The latter is characterized by the synthesis and secretion of an iron-chelating substance phytosiderophore (Takagi, 1976) and by specific uptake of the Fe(III)–phytosiderophore complex. Since the structure of the first phytosiderophore (mugineic acid, MA) was first identified in barley by Takemoto et al. (1978), nine analogs have been isolated and identified from various graminaceous species and cultivars (Ma, 2005). Compared to siderophores secreted from micro-organisms [molecular weight (MW) 500–1000] (Neilands, 1981), the known phytosiderophores have small molecular sizes (MW 294–336) and quite different structures. All of them contain six functional groups, which play a crucial role in the specific chelation and uptake of iron.

The process of iron acquisition by strategy II plants can be divided into four main steps: (i) biosynthesis of phytosiderophores inside the roots; (ii) secretion of phytosiderophores to the rhizosphere; (iii) solubilization of insoluble Fe(III) in soils by chelation of phytosiderophores, and (iv) uptake of phytosiderophore–Fe(III) complexes by the roots. The biosynthetic pathway of phytosiderophores has been elucidated (Ma and Nomoto, 1996). l-Methionine (Met) serves as a precursor for all known phytosiderophores (Ma and Nomoto, 1992, 1993, 1994; Mori and Nishizawa, 1987; Shojima et al., 1990), and the biosynthesis of phytosiderophores is associated with the Met cycle (Ma et al., 1995). All phytosiderophores share the same pathway from l-Met to 2′-deoxymugineic acid (DMA) via nicotianamine (NA), but subsequent steps are different among plant species and cultivars. Most genes involved in phytosiderophore synthesis have been cloned, including APRT (adeninephosphoribosyl transferase), SAMS (S-adenosylmethionine synthetase), NAS (NA synthase), NAAT (NA aminotransferase) and iron-deficiency-specific clones IDS2 and IDS3, deoxigenases that hydroxylate the C-3 and C-2′ positions of MA (Mori, 1999). All of these genes are induced specifically in the roots under conditions of iron deficiency. Recently, two cis-acting elements, IDE1 and IDE2, of the barley IDS2 gene promoter for iron-deficiency-inducible and root-specific expression were identified (Kobayashi et al., 2003).

The secretion of phytosiderophores shows a distinct circadian rhythm (Takagi et al., 1984). The secretion time is likely to be controlled by temperature around the roots (Ma et al., 2003). It has reported that MAs are secreted probably via anion channels using the potassium gradient between the cytoplasm and the cell exterior, as suggested by the decrease in MA secretion upon treatment with anion channel blockers or the potassium gradient inhibitor valinomycin (Sakaguchi et al., 1999). On the other hand, an increase in the size and number of particular vesicles, which are thought to originate from the rough endoplasmic reticulum (rER), were observed in root epidermal cells of iron-deficient barley prior to the onset of secretion of MAs (Negishi et al., 2002). Following secretion, phytosiderophores solubilize soil iron [e.g. Fe(OH)3] by efficient chelation in soils (Ma and Nomoto, 1996).

The last step of iron acquisition is the transport of iron into the root cells. Previous physiological studies have shown that, in contrast to the uptake mechanism in strategy I plants, the Fe(III)–phytosiderophore complex is taken up across the plasma membrane of root cortex cells as an undissociated molecule in strategy II plants (Römheld and Marschner, 1986). A gene yellow stripe 1 (YS1) that is necessary for the uptake of Fe(III)–phytosiderophore has been identified in maize (Curie et al., 2001). The protein (ZmYS1) is a highly hydrophobic protein with 12 putative transmembrane-spanning domains. ZmYS1 functionally complements yeast strains that are defective in iron uptake on media containing Fe(III)–phytosiderophores, but not on media containing Fe(III) citrate, suggesting that ZmYS1 is a phytosiderophore-dependent iron transporter (Curie et al., 2001). Further characterization shows that ZmYS1 is an H+–Fe(III)–phytosiderophore co-transporter (Schaaf et al., 2004). However, this transporter has a broad specificity for both metals and ligands. The heterologous expressions of ZmYS1 in yeast and Xenopus oocytes show that the protein can transport phytosiderophore-bound metals including zinc, copper and nickel (Roberts et al., 2004; Schaaf et al., 2004). It also has transport activity for nickel, Fe(II) and Fe(III) complexes with NA (Schaaf et al., 2004). However, a structural study with barley shows that the uptake system has a very strict specificity for both metals and ligands; neither copper, zinc nor cobalt in complex with various phytosiderophores is taken up, nor is iron in complex with other ligands including NA (Ma et al., 1993). Such difference in substrate specificity between ZmYS1 as studied with yeast/oocytes and the possible counterpart in barley led us to speculate that a different transporter for the specific Fe(III)–phytosiderophore complex is present in barley roots.

In the present study, a gene encoding an iron–phytosiderophore transporter (HvYS1) was cloned from barley roots under iron-deficient conditions, and its high specificity determined in terms of its localization and the metal and phytosiderophore utilized as substrates in comparison with ZmYS1.

Results

Identification of the HvYS1 cDNA in barley

To isolate a gene encoding the iron–phytosiderophore transporter (HvYS1) in barley roots, a cDNA library was constructed from Fe-deficient roots. We found four barley EST clones (AF472629, BJ47081, BJ448359 and BQ765689) in the DNA Data Bank of Japan (DDBJ) database based on the amino acid sequence of ZmYS1 (Curie et al., 2001). 5′- and 3′-rapid amplification of cDNA ends (RACE) using specific primers in these ESTs resulted in the isolation of a full-length cDNA from the cDNA library. The cDNA is 2430 bp long, and the deduced polypeptide is 678 amino acids (Figure 1a). This gene is predicted to encode a plasma membrane protein (Figure 1b), and a blast search shows that HvYS1 belongs to a member of the oligopeptide transporter (OPT) family, which transports oligopeptides in several organisms, including bacteria, archaea, fungi and plants (Yen et al., 2001). HvYS1 is the closest homolog to ZmYS1 known so far, with 72.7% identity and 95.0% similarity (Figure 1a,c). In particular, all predicted transmembrane regions of the two proteins have high similarities (Figure 1a). The N-terminal domain of HvYS1 has an acidic amino acid-rich sequence (seven glutamic and seven aspartic acid residues out of 49 amino acids, 29%), as does the N-terminus of ZmYS1 (11 glutamic and five aspartic acid residues out of 52 amino acids, 31%).

Figure 1.

 Sequence analyses of HvYS1, an iron-phytosiderophore transporter in barley.
(a) Comparison of the putative amino acid sequences of HvYS1 (Hordeum vulgare L.) with ZmYS1 (Zea mays yellow stripe 1, AF186234; Curie et al., 2001). HvYS1 shows high homology to ZmYS1 with 72.7% identity (in black) and 95.0% similarity (in gray). Putative transmembrane regions (HvYS1, I–XI; ZmYS1, I–XII) are indicated by lines.
(b) Predicted membrane-spanning structure of HvYS1 by the SOSUI program (Hirokawa et al., 1998).
(c) Phylogenetic relationship of YS1-like proteins in rice (blue), Arabidopsis (black) and maize (green).

Expression and localization of HvYS1

The expression pattern of HvYS1 gene in barley was investigated in different tissues including roots, stem, mature leaves and young leaves using real-time RT-PCR. The results showed that the HvYS1 gene was mainly expressed in the roots (Figure 2). Furthermore, the expression was enhanced 50-fold in the Fe-deficient roots compared to the Fe-sufficient roots (Figure 2).

Figure 2.

 Real-time RT-PCR analysis of HvYS1 expression.
Total RNA from roots (R), old (OL) or young chlorotic leaves (YL), and stems (S) from barley grown under Fe-sufficient (+Fe) or Fe-deficient (−Fe) condition was reverse-transcribed and amplified by PCR. The cDNA was amplified using primers specific to HvYS1 or to GAPDH as an internal control.

We investigated the localization of the HvYS1 expression to examine its physiological role. In situ hybridization analysis of iron-deficient barley roots indicated that HvYS1 mRNA was mainly present in epidermal root cells on hybridization with an antisense probe (Figure 3b,d), whereas no signal was observed with an HvYS1 sense probe (Figure 3a,c).

Figure 3.

 Expression of HvYS1 transcript in barley roots.
Paraffin-embedded longitudinal sections (a, b) and cross-sections (c, d) (5 μm) from barley roots under Fe-deficient conditions were hybridized with sense (a, c) and antisense (b, d) probes labeled by digoxigenin (DIG). Scale bar, 100 μm.

The subcellular localization of HvYS1 was first examined by transient transformation of HvYS1 fused with green fluorescent protein (GFP) into onion epidermal cells. This fusion protein was expressed on the plasma membrane in the cells, whereas the signal was also observed in the cytoplasm and nuclei of cells expressing GFP alone (Figure 4). This results show that HvYS1 is a transporter that is localized in the plasma membrane.

Figure 4.

 Subcellular localization of HvYS1–GFP fusion and GFP proteins in onion epidermal cells.
HvYS1:GFP shows localization to the plasma membrane. Cell walls were counter-stained with propidium iodide. Scale bar, 100 μm.

The localization of HvYS1 protein was further examined by means of rabbit anti-HvYS1 polyclonal antibody staining. Signal was observed on the plasma membranes of epidermal cells and was greatly enhanced under iron deficiency (Figure 5). To examine the specificity of the antibody used, the antibody was pre-incubated with the peptide epitope before staining. As a result, a strong signal in the epidermal cells disappeared (Figure 5), suggesting that the antibody has a high specificity for HvYS1. These results are in agreement with those of the RT-PCR (Figure 2) and in situ hybridization (Figure 3).

Figure 5.

 Localization of the HvYS1 protein in the roots of barley grown under Fe-sufficient (+Fe) and -deficient (−Fe) conditions.
Immunostaining was performed using anti-HvYS1 antibody. The specificity of the antibody was tested by pre-incubating the antibody with the epitope peptide [−Fe (+peptide)]. Scale bar, 50 μm.

Transport activity and substrate specificity of HvYS1

The transport activity and substrate specificity of HvYS1 were investigated and compared with those of ZmYS1 using heterologous expression in yeast. We individually subcloned HvYS1 and ZmYS1 cDNAs into the yeast expression vector pFL61 and then transformed the cDNAs into the DDY4 strain, a double yeast mutant (fet3fet4) that cannot grow on iron-limited medium (Dix et al., 1997). When Fe(III)–MA was supplied as the sole iron source, both HvYS1 and ZmYS1 expression restored growth of the fet3fet4 mutant (Figure 6). In the presence of Fe(II)–NA, however, while ZmYS1 expression allowed growth of the mutant (Roberts et al., 2004; Schaaf et al., 2004), HvYS1 did not (Figure 6). The possibility of Fe(II) transport by HvYS1 was excluded by the unaffected growth of the fet3fet4 mutant in Fe(III)–MA medium supplemented with 4,7-biphenyl-1,10-phenanthroline-disulphonic acid (BPDS), a strong Fe(II) chelator (Figure 6).

Figure 6.

 Functional complementation of yeast.
Plasmids expressing HvYS1, ZmYS1 or empty vector (VEC) were individually introduced into the iron uptake-defective yeast strain DDY4 (fet3fet4; Dix et al., 1997). Yeast was grown on minimum medium lacking uracil supplemented with 10 μm Fe(III)–MA, 10 μm Fe(II)–NA, 10 μm Fe(III)–MA + 10 μm BPDS. Three yeast cell dilutions (adjusted optional densities at 600 nm to 0.2, 0.02, and 0.002) were spotted onto the plates.

We further examined the substrate specificity of HvYS1 for protein heterologously expressed in Xenopus laevis oocytes (Figure 7). Oocytes were voltage-clamped at −60 mV in buffer solution at pH 7.6 and then superfused with buffer containing various metal–MA and Fe(II)–NA complexes. In oocytes injected with the cRNA encoding HvYS1, Fe(III)–MA induced currents in a voltage-dependent manner (Figure 7a). Currents were absent in water-injected (Figure 7b,c) or non-injected control oocytes (data not shown). In oocytes injected with the cRNA encoding HvYS1, currents were induced by Fe(III)–MA, but not by MA in complex with other metals, including copper, zinc, nickel, manganese and cobalt, or by Fe(II)–NA. In contrast, in oocytes injected with ZmYS1 cRNA, currents were induced not only by Fe(III)–MA, but also by Cu(II)–MA and Zn(II)–MA similarly, and Ni(II)–MA, Mn(II)–MA and Co(II)–MA to a lesser extent (Figure 7c). Fe(II)–NA also induced similar currents to Fe(III)–MA.

Figure 7.

 Transport activity of HvYS1 and ZmYS1 by two-electrode voltage clamp analysis in Xenopus laevis oocytes.
Between three and six individual oocytes injected with HvYS1 or ZmYS1 cRNA were incubated for 2–4 days.
(a) Voltage dependence of Fe(III)–MA-induced currents in HvYS1-expressing oocytes in 50 μm Fe(III)–MA (pH 7.6).
(b) Currents induced by various metal complexes at 50 μm in oocytes expressing HvYS1.
(c) Currents induced by various metal complexes at 50 μm in oocytes expressing ZmYS1. Measurement was carried out at −60 mV. Relative currents to Fe(III)–MA are shown. Error bars show standard deviation (n = 3–6).

Discussion

The amount of phytosiderophore secretion is roughly parallel to the levels of tolerance of the plant to iron deficiency, and generally follows the order: barley/wheat >oat/rye > maize/sorghum > rice (Kawai et al., 1988; Marschner et al., 1986). In the present study, a gene encoding iron–phytosiderophore transporter (HvYS1) was cloned and characterized from barley roots, which are the most tolerant to iron deficiency. This gene, which shows very close homology to ZmYS1 (Figure 1c), encodes a transporter with quite different tissue localization and specificities. HvYS1 was only expressed in the root (Figure 2), while ZmYS1 was expressed in both the roots and leaves of maize (Curie et al., 2001), although the expression of both HvYS1 and ZmYS1 was greatly enhanced under iron deficiency (Figure 2). The different localization between HvYS1 and ZmYS1 suggests that HvYS1 has some functions that differ from those of ZmYS1. The detailed cellular localization of ZmYS1 has not been investigated in maize roots, but our results from in situ hybridization and antibody staining clearly show that both the mRNA of HvYS1 and its encoded protein are localized in the epidermal cells (Figure 3 and 5). This localization is similar to that of FRO2 and IRT1, which are involved in reduction of ferric iron on the plasma membrane and the subsequent transport of ferrous iron, respectively, in strategy I plants (Eide et al., 1996; Robinson et al., 1999). Both IRT1 and FRO2 are reported to be localized in the epidermal cells of Fe-deficient Arabidopsis roots (Connolly et al., 2003; Vert et al., 2002). The localization of HvYS1 suggests that HvYS1 is a transporter that is responsible for primary uptake of the iron–phytosiderophore complex in barley roots, while ZmYS1 may be also involved in the internal transport of iron.

The specificity for metal complexes also was greatly different between HvYS1 and ZmYS1. Both yeast complementation and oocyte experiments clearly showed that HvYS1 has high substrate specificity; only the Fe(III)–phytosiderophore complex could be transported by HvYS1 (Figure 6 and 7). In contrast, our results (Figure 7) and several other studies have shown that ZmYS1 transports various phytosiderophore-bound metals including zinc, copper and nickel (Roberts et al., 2004; Schaaf et al., 2004). It also transports Ni(II), Fe(II) and Fe(III) complexes with NA. The high substrate specificity of HvYS1 agrees well with the strict recognition of the Fe(III)–phytosiderophore complex observed in a previous physiological study with barley plants (Ma et al., 1993). The difference in the substrate specificity between HvYS1 and ZmYS1 may result from the stereostructure of the transport protein. It has been predicted on the basis of the sosui program (Hirokawa et al., 1998) that HvYS1 has 11 putative transmembrane-spanning domains (Figure 1b) while ZmYS1 has 12 putative transmembrane-spanning domains (Curie et al., 2001).

Comparison of the amino acid sequence between HvYS1 and ZmYS1 shows that the N-terminal domains (approximately 49 residues) and the loop between the fifth and sixth predicted transmembrane regions of HvYS1 have low similarity (Figure 1a). These regions may be responsible for different substrate recognition, although further studies are needed.

In spite of low sequence similarity, the N-terminal domains of both HvYS1 and ZmYS1 share the basic feature of being rich in glutamic and aspartic acid residues. Both protein also have in this region EXXE sequences, known as an Fe3+-binding motif in a signal transduction system that responds to extracellular iron (Wösten et al., 2000). These domains of HvYS1 and ZmYS1 are speculated to be involved in recognition of the Fe(III)–phytosiderophore complex.

Eight genes in Arabidopsis (Curie et al., 2001), 18 genes in rice (Gross et al., 2003; Koike et al., 2004), and four ESTs in maize (Curie et al., 2001) that have similarities to ZmYS1 (Curie et al., 2001) were identified in database searches (Figure 1c). Recent studies have shown that most of them may function in the intracellular transport, translocation, and distribution of iron and other metals as NA complexes in various plant tissues. For example, Arabidopsis YSL2 (AtYSL2), one of the closest homologs to ZmYS1, reportedly transports both Fe(II) and copper in complex with NA (DiDonato et al., 2004), although contrary results also have been reported (Schaaf et al., 2005). Recently, Arabidopsis YSL1 (AtYSL1) has been implicated in the long-distance circulation of iron and NA and their delivery to the seed (Jean et al., 2005). Furthermore, it has been reported that rice YSL2 (OsYSL2 in Figure 1c) transports Fe(II) and manganese(II)–NA complexes, but not Fe(III)–phytosiderophore, and is expressed in the phloem cells of the vascular bundles (Koike et al., 2004). These results suggest that OsYSL2 is a metal–NA transporter responsible for the phloem transport of iron and manganese in rice.

Iron deficiency is particularly pronounced in plants grown on calcareous soils, not only because of the high pH levels, but also because of other factors such as high calcium and magnesium concentrations and high bicarbonate levels, which reduce the availability and uptake of iron (Ma and Nomoto, 1996; Mino et al., 1983). From an ecological point of view, graminaceous plants obviously can adapt to calcareous soils better than other types of plants (Römheld, 1991). This is because their uptake systems function even at a high pH, elevated bicarbonate concentrations, and in the presence of excessive Ca2+ and Mg2+. Therefore, to overcome iron-deficiency stress problems, several molecular targets involved in iron transport by strategy II are indicated. It has been reported that a barley genomic fragment containing the NaaA and NaaB genes encoding NAAT(Takahashi et al., 1999), upon introduction into rice, resulted in increased DMA secretion and consequently conferred on rice an enhanced tolerance to low iron availability (Takahashi et al., 2001). As HvYS1 is an Fe(III)–phytosiderophore transporter for direct uptake of iron from alkaline soil, HvYS1 combined with NaaA and NaaB genes could be more promising for transgenic plants.

In conclusion, this identification of the transporter HvYS1 specific for the uptake of Fe(III)–phytosiderophore will provide better understanding of the iron acquisition mechanism in graminaceous plants and help to produce plants with enhanced tolerance to iron-deficiency stress.

Experimental procedures

Plant growth

Barley plants were grown in 1/5 strength Hoagland nutrient solution (pH 5.6) containing macronutrients KNO3 (1 mm), Ca(NO3)2 (1 mm), MgSO4 (0.4 mm), (NH4)H2PO4 (0.2 mm), and micronutrients H3BO3 (3 μm), MnCl2 (0.5 μm), CuSO4 (0.2 μm), ZnSO4 (0.4 μm) and (NH4)6Mo7O24 (1 μm), with or without Fe(III)–MA (20 μm). The nutrient solution was aerated and renewed every 2 days. MA was purified from root exudates of Fe-deficient barley (Takagi et al., 1984). The Fe(III)–MA complex was prepared by mixing equimolar amounts of MA and FeCl3. The pH of the complex was adjusted to 5.6 with 1 n NaOH.

RNA extraction and cDNA cloning

RNA was extracted from barley roots that had been subjected to iron deficiency [without Fe(III)–MA] for 7 days by means of the ConcertTM Plant RNA Reagent (Invitrogen, Carisbad, CA, USA). HvYS1 cDNA (2430 bp) was identified from RNA by 5′- and 3′-RACE systems (Invitrogen and Roche Diagnostics, Penzberg, Germany) using gene-specific primers (GSPs) designed from barley ESTs AF472629, BJ4708221, BJ448359, BQ765689 by a homology search using the ZmYS1 sequence (AF186234; Curie et al., 2001). 5′-RACE with primers 5′-CCACAAGCATCGCCTCCAG-3′ (GSP1), 5′-CATCGCCTCCAGTGTAGAACC-3′ (GSP2) and 5′-CAGTGTAGAACCATTGGAAG-3′ (GSP3; for the Invitrogen kit), and 5′- GAATAGCAGTTGCAGTCC-3′ (GSP1′), 5′-GTAGTCGACGACCAGTACCTG-3′ (GSP2′) and 5′-CGACCAGTACCTGTCTCAGG-3′ (GSP3′; for the Roche Diagnostics kit), and 3′-RACE with primers 5′-CATTGCCGGCCTTGTTGCTG-3′(GSP) and 5′-CGGCCTTGTTGCTGGCACC-3′(nest GSP) were performed with Taqex polymerase (Takara, Shiga, Japan) and a thermal cycler (model GeneAmp PCR system 9700; Applied Biosystems, Foster City, CA, USA). The resulting PCR products were purified by use of a Qiaquick Gel Extraction kit (Qiagen, Inc., Valencia, CA, USA) and then subcloned into the pCRII-TOPO vector by a TA cloning kit (Invitrogen). Subcloned inserts were sequenced on an ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems) by means of a Big-Dye sequencing kit (Applied Biosystems) and universal primers (M13 forward and reverse).

Real-time RT-PCR

Six days after treatment with or without Fe(III)–MA, seedlings were sectioned into root tips, mature leaves, young leaves and stems. Each plant part was frozen immediately in liquid nitrogen and stored at −80°C until use. Frozen samples were ground in a mortar in liquid nitrogen, then total RNA was isolated by means of an RNeasy plant mini kit (Qiagen). Total RNAs were converted to cDNAs by a SuperScriptTMfirst-strand synthesis system for RT-PCR (Invitrogen) in a thermal cycler (PTC-100 Programmable Thermal Controller; Bio-Rad, Hercules, CA, USA). cDNAs of HvYS1 and GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase as the loading control, were amplified by SYBR premix Ex taqTM (Takara) and real-time RT-PCR (ABI Prism 7000 sequence detection system; Applied Biosystems) with primer pairs 5′-AAAAAATGCGGACGACACTGT-3′ (forward) and 5′-AGGCATAACCAGCGTATGCC-3′ (reverse) for HvYS1, and 5′- TGCCATGACTGCTACCCAGA-3′ (forward) and 5′- CACCAGTGCTGCTTGGAATG-3′ (reverse) for GAPDH.

In situ hybridization

To prepare the RNA probes, the ORF region of HvYS1 cDNA was amplified by PCR with the forward primer 5′-GCTCTAGAATGGACATCGTCGCC-3′ and reverse primer 5′-CCCAAGCTTTTAGGCAGCAGGTAG-3′, and then inserted into the XbaI, HindIII site of pBluescript II KS(+) vector (Stratagene, La Jolla, CA, USA). The plasmid was linearized with XbaI (sense strand) and HindIII (antisense strand). Digoxigenin-(DIG)-11-UTP-labeled, single-stranded sense and antisense RNA probes were prepared with a DIG RNA labeling mixture, and the corresponding T3 or T7 RNA polymerase (T3 RNA polymerase as the antisense probe and T7 RNA polymerase as the sense probe, Roche Diagnostic). The probes were degraded to a mean length of 150 bp by incubation in alkali at 60°C. In situ hybridization was done with 5 μm paraffin sections of iron-deficient and -sufficient barley roots as described previously (Jackson, 1991). Sections were hybridized at 50°C for 17 h. After being washed at 55°C and treated with anti-DIG alkaline phosphatase (Roche Diagnostics) for 1 h at room temperature, sections were stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl (NBT/BCIP; Nakarai tesque, Kyoto, Japan).

Subcellular localization of HvYS1:GFP

Transient transformation of onion epidermal cells was performed with a biolistic PDS-1000/He Particle Delivery System (Bio-Rad). Gold particles, diameter 1 μm, were coated with plasmid DNA carrying a fusion gene of HvYS1 and GFP or GFP only, and prepared for bombardment according to the manufacturer's protocol. The pressure was 9 MPa. Bombarded cells were kept in the dark at room temperature for 20 h. Fluorescence was observed with a laser-scanning confocal microscope (LSM510; Karl Zeiss, Jena, Germany). Cell walls were counter-stained with propidium iodide. Plasmolysis was induced with 1 m mannitol.

Immunohistology

The synthetic peptide C-TRIAPEIDRDEALE (positions 9–22 of HvYS1) was used to immunize rabbits to obtain antibodies against HvYS1. Iron-sufficient and -deficient barley roots, prepared as described above, were fixed in 4% w/v paraformaldehyde and 60 mm sucrose then buffered with 50 mm cacodylic acid (pH 7.4) at room temperature for 2 h with occasional degassing. After three washes with 60 mm sucrose and 50 mm cacodylic acid (pH 7.4), the fixed samples were embedded in 5% agar and sectioned 80-μm thick with a microslicer (ZERO 1; Dosaka EM, Kyoto, Japan). The sections were placed on microscope slides, incubated with PBS (10 mm phosphate-buffered saline, pH 7.4, 138 mm NaCl, 2.7 mm KCl) containing 0.1% w/v pectolyase Y-23 (Seishin, Tokyo, Japan) at 30°C for 2 h then re-incubated in PBS containing 0.3% v/v Triton X-100 at 30°C for 2 h, washed three times with PBS and blocked with 5% w/v BSA in PBS. The slides were incubated at 37°C overnight in a humid chamber with the purified rabbit anti-HvYS1 polyclonal antibodies (1:50 dilution in PBS). After three washes in PBS and blocking with 5% w/v BSA in PBS, the slides were exposed to secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG; Molecular Probes, Eugene, OR, USA) for 2 h at room temperature, washed five times in PBS, and mounted with 50% v/v glycerol in PBS. Samples were examined with a laser scanning confocal microscope (LSM510; Karl Zeiss). To check the specificity, the antibody (1:50 dilution) was pre-incubated with the epitope peptide used for preparation of antibody at 25 nmol ml−1 for 1 h at room temperature before staining as described above.

Yeast functional complementation

Saccharomyces cerevisiae strain DY1457 (MATaade6can1his3leu2trp1ura3) and the iron-uptake-defective strain DDY4 (MATaade6can1his3leu2trp1ura3fet3-2::HIS3fet4-1::LEU2; Dix et al., 1997) were transformed with HvYS1 and ZmYS1 plasmids individually cloned into the NotI-digested pFL61 vector (ATCC, Manassas, VA, USA). Yeast functional complementation assays were performed as previously described (Roberts et al., 2004). All plate assays were carried out on a metal-free yeast nitrogen base (lacking the metal nutrients Cu, Zn, Mn and Fe; US Biological, Swampscott, MA, USA). Yeast were grown on synthetic defined (SD) media lacking uracil but supplemented with 10 μm Fe(III)–MA or Fe(II)–NA and in the presence or absence of 10 μm 4,7-biphenyl-1,10-phenanthroline-disulphonic acid (BPDS; Dojin, Kumamoto, Japan). After spotting at three yeast cell dilutions (optical densities at 600 nm of 0.2, 0.02 and 0.002), plates were incubated for 3 days at 30°C. MA was purified from root exudates of Fe-deficient barley. NA was purchased from Hasegawa Co. (Kawasaki, Japan).

Electrophysiological studies in Xenopus laevis oocytes

The open reading frame (ORF) region of HvYS1 cDNA was amplified by PCR with the forward primer 5′-GCTCTAGACCACCATGGACATCG-3′ and reverse primer 5′-CGCGGGATCCTTAGGCAGCAGGTAG-3′, and for ZmYS1 with the forward primer 5′-GCTCTAGACCACCATGGACCTTG-3′ and reverse primer 5′-CGCGGGATCCCTAGCTTCCAGGAGTGAA-3′, then inserted into the XbaI, BamHI site of the Xenopus expression vector pSP64 polyA (Promega, Madison, WI, USA). The plasmid was linearized with BamHI, and cRNA was transcribed in vitro with SP6 RNA polymerase (mMESSAGE mMACHINE kit; Ambion, Austin, TX, USA). The cRNA solution (50 nl, 0.05 μμl−1) was injected into Xenopus oocytes which then were incubated for 2–4 days at 16°C in ND96 buffer (pH 7.6) containing NaCl (96 mm), KCl (2 mm), CaCl2 (1.8 mm), MgCl2 (1 mm) and HEPES (5 mm). The oocytes were voltage-clamped (Oocite clamp OC-725c, Warner Instrument, Hamden, CT, USA), and steady-state currents obtained in response to addition of the metal–chelate complex (10 μl, 7.5 mm, final concentration; 50 μm). Transport activity was analysed with Origin 6.1 software (Microcal Software, Tokyo, Japan).

Acknowledgements

We are grateful to David Eide for his kind gift of the fet3fet4 yeast mutant, Elsbeth L. Walker for providing the ZmYS1 cDNA, Haruyo Hatanaka, Eiichiro Ono, Honoo Satake, Tsuyoshi Kawada and Atsuhiro Kanda for their invaluable technical advices, and to Shoichi Kusumoto for his encouragement and discussion. This study was supported in part by a CREST/JST (Japan Science and Technology Cooperation) grant to J. F. Ma.

The full-length cDNA sequence of HvYS1 has been deposited in the DNA Data Bank of Japan (DDBJ) under the accession number AB214183.

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