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First published online November 14, 2008; 10.1104/pp.108.128132 Plant Physiology 149:297-305 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
OsFRDL1 Is a Citrate Transporter Required for Efficient Translocation of Iron in Rice1,[OA]Research Institute for Bioresources, Okayama University, Kurashiki 710–0046, Japan
Multidrug and toxic compound extrusion (MATE) transporters represent a large family in plants, but their functions are poorly understood. Here, we report the function of a rice (Oryza sativa) MATE gene (Os03g0216700, OsFRDL1), the closest homolog of barley (Hordeum vulgare) HvAACT1 (aluminum [Al]-activated citrate transporter 1), in terms of metal stress (iron [Fe] deficiency and Al toxicity). This gene was mainly expressed in the roots and the expression level was not affected by either Fe deficiency or Al toxicity. Knockout of this gene resulted in leaf chlorosis, lower leaf Fe concentration, higher accumulation of zinc and manganese concentration in the leaves, and precipitation of Fe in the root's stele. The concentration of citrate and ferric iron in the xylem sap was lower in the knockout line compared to the wild-type rice. Heterologous expression of OsFRDL1 in Xenopus oocytes showed transport activity for citrate. Immunostaining showed that OsFRDL1 was localized at the pericycle cells of the roots. On the other hand, there was no difference in the Al-induced secretion of citrate from the roots between the knockout line and the wild-type rice. Taken together, our results indicate that OsFRDL1 is a citrate transporter localized at the pericycle cells, which is necessary for efficient translocation of Fe to the shoot as a Fe-citrate complex.
Multidrug and toxic compound extrusion (MATE) proteins are widely present in bacteria, fungi, plants, and mammals (Omote et al., 2007  ). These proteins are characterized by having 400 to 700 amino acids with 12 transmembrane helices. Although no apparent consensus sequence is conserved in all MATE proteins, they share approximately 40% sequence similarity. MATE protein was first identified as a multidrug transporter for norfloxacin and ethidium bromide in Vibrio parahaemolyticus and Escherichia coli (Morita et al., 1998), but subsequent studies have shown that the MATE proteins transport various organic substrates, such as alkaloids, berberine, and acriflavine (Omote et al., 2007). Therefore, MATE-type transporters are proposed to function as fundamental molecular machinery for the excretion of metabolic and xenobiotic organic compounds from the body.
Plants have a higher diversity of MATE-type transporters than bacteria and animals (Omote et al., 2007
Recently, three studies have reported that some MATE proteins are involved in the transport of citrate, which is required for iron (Fe) translocation or aluminum (Al) detoxification. FRD3 from Arabidopsis has been demonstrated to be a citrate transporter, which is required for Fe translocation from the roots to the shoots (Durrett et al., 2007
Phenotype of OsFRDL1 Knockout Line
OsFRDL1 (Os03g0216700) was initially isolated as a homolog of AtFRD3 (Fig. 1; Inoue et al., 2004
Because AtFRD3 has been implicated in the Fe translocation in Arabidopsis (Green and Rogers, 2004 ), we first investigated the involvement of OsFRDL1 in Fe nutrition by growing the plants at low and high Fe concentrations. When the wild-type rice and two Tos-17 insertion lines were grown at 10 µM Fe (as FeSO4), no visible difference was observed among the three lines (Fig. 3A
). However, at 0.2 µM Fe, chlorosis was observed in the newly expanded leaves of two Tos-17 lines, but not in the wild type (Fig. 3B).
The Fe3+ precipitation in the roots was investigated at 10 µM Fe with Perls blue staining (Green and Rogers, 2004 ). Fe precipitation was observed in the epidermal cells of the roots of all three lines (Fig. 3, C–E), which was not observed in Arabidopsis roots (Green and Rogers, 2004). This distinct precipitation in rice is attributed to secreted oxygen from rice roots, which oxidizes ferrous iron (Fe2+) into insoluble ferric iron (Fe3+) on the root surface (Horiguchi, 1995). However, there was no difference in the epidermal staining among three lines (Fig. 3, C–E). In contrast, heavy staining was observed in the central vascular part of the knockout line, ND (Fig. 3, D and G), but not in the wild-type line (Fig. 3, C and F). In the knockdown line NC, staining was also observed in the central vascular part (Fig. 3, E and H), although the intensity was not as strong as that of ND. We further investigated the link between Fe accumulation detected with Perls blue staining in the root stele and Tos-17 insertion using heterozygous progeny of ND. Genotyping analysis with specific primers in 50 seedlings showed that wild-type, heterozygotes, and homozygotes segregated at 10:27:13, respectively. All 13 mutant homozygote seedlings showed heavy staining in the central vascular part. These results demonstrate that the phenotypes observed in ND are caused by loss of function of OsFRDL1. We then used ND, which shows clearer phenotypes, for further investigations.
We compared the Fe concentration in the roots and shoots between the wild-type rice and the knockout line (ND). The concentration of Fe in the shoots was significantly lower in the knockout line than in the wild-type rice at either Fe concentration (Fig. 4A ). The shoot Fe concentration of ND was 77% and 53.5% of the wild-type rice, respectively, at 0.2 and 10 µM. The shoot Fe concentration of ND at 10 µM Fe was higher than that of wild type at 0.2 µM. This is in agreement with the phenotype of ND at 10 µM Fe (no chlorosis; Fig. 3A). By contrast, the concentration of Fe in the roots was 2 times higher in the knockout line than in the wild-type line at 0.2 µM Fe (Fig. 4B). At 10 µM Fe, the root Fe increased to an extremely high concentration, probably due to precipitation of Fe on the root epidermal layer (Fig. 4C). The root Fe concentration is also slightly higher in ND, but there was no significant difference between ND and the wild-type rice. These results indicate that knockout of OsFRDL1 causes accumulation of Fe in the roots and decreased Fe concentration in the shoots.
Analysis of other metals showed that the shoot concentration of zinc (Zn) and manganese (Mn) was 15% higher in ND than in wild type at 0.2 µM Fe, but there was no difference between the two lines at 10 µM Fe (data not shown). There was no difference in the shoot copper (Cu) concentration between ND and wild type at either Fe concentrations. Because Fe deficiency is more pronounced in the ND knockout line, higher concentrations of Zn and Mn in the ND shoots at lower Fe concentration probably resulted from Fe deficiency-enhanced expression of IRT1, a Fe2+ transporter (Eide et al., 1996 ). IRT1 has been shown to transport other divalent metals, such as Zn and Mn, in addition to Fe (Eide et al., 1996; Korshunova et al., 1999). In fact, the expression of OsIRT1 was markedly enhanced in ND roots at 0.2 µM Fe (data not shown). Overall, the shoot concentration of Zn, Mn, and Cu was higher at 0.2 µM Fe than that at 10 µM Fe. This is probably due to lower expression of IRT1 at a higher Fe concentration.
Accumulation of Fe in the roots and decreased Fe concentration in the shoots of the knockout line suggest that OsFRDL1 is involved in the translocation of Fe from the roots to the shoots. Fe was reported to be translocated in the form of a Fe-citrate complex in xylem sap (Tiffin, 1970
Expression Pattern of OsFRDL1 and Localization of OsFRDL1 The expression of OsFRDL1 was examined with quantitative reverse transcription (RT)-PCR. OsFRDL1 was mainly expressed in the roots, but not in the shoots (Fig. 6A ). A time course experiment showed that the expression in the roots was not affected by Fe deficiency (Fig. 6, A and B), in contrast to IRT1, which expression was greatly increased with development of Fe deficiency (Fig. 6C). Interestingly, the expression of OsFRDL1 was higher in the mature root zone than in the root tip (Fig. 6D). This result is in agreement with Fe precipitation and development of xylem.
The localization of OsFRDL1 was examined with an anti-OsFRDL1 antibody. Immunostaining showed that OsFRDL1 was localized at pericycle cells (Fig. 7, A and B ). This result is consistent with OsFRDL1 promoter GUS staining (Inoue et al., 2004 ). No signal was observed in the knockout line (Fig. 7C), indicating the high specificity of the anti-OsFRDL1 antibody. The subcellular localization of OsFRDL1 was found to be localized on the plasma membrane by using heterologous expression in the onion (Allium cepa) epidermal cells (data not shown), which was the same as that reported previously (Inoue et al., 2004).
OsFRDL1 Was Not Involved in the Al-Induced Secretion of Citrate
Rice secretes citrate from the roots in response to Al, although the amount secreted is small (Ma et al., 2002
OsFRDL1 Is Able to Transport Citrate in a Heterologous Expression System The above results suggest that OsFRDL1 encodes a transporter for citrate at the plasma membrane of pericycle cells, which is required for efficient translocation of Fe3+ as a citrate complex from the roots to the shoots. To confirm whether OsFRDL1 has activity to transport citrate, we expressed OsFRDL1 in Xenopus oocytes. The efflux activity for citrate was significantly higher in the oocytes injected with OsFRDL1 cRNA than in oocytes injected with water (Fig. 9 ). This result indicates that OsFRDL1 is able to transport citrate out of the cells.
OsFRDL1 has 87% sequence homology with HvAACT1 and shares 57% sequence identity with AtFRD3 at the amino acid level (Fig. 1). However, our results indicate that OsFRDL1 is not involved in the Al-induced secretion of citrate like HvAACT1, but in Fe translocation like AtFRD3. OsFRDL1, HvAACT1, and AtFRD3 are from different plant species, but all show efflux activity for citrate when they were expressed in heterologous systems (Fig. 9; Durrett et al., 2007 ; Furukawa et al., 2007), indicating that they are all efflux transporters of citrate. Since the expression of OsFRDL1 was not responsive to Al (Fig. 8B), their different roles may be attributed to different tissue-specific localization. HvAACT1 is localized on the plasma membrane of the root epidermal cells, while AtFRD3 and OsFRDL1 are on the pericycle cells (Fig. 7; Green and Rogers, 2004). It is likely that activation of HvAACT1 by Al is required to transport citrate out of the cells (Furukawa et al., 2007), but Al usually stops at the epidermal cells and outer cortex cells (Ma et al., 2004). Therefore, Al may not reach the pericycle cells to activate OsFRDL1. This is supported by the finding that citrate secretion occurred when AtFRD3 was overexpressed in Arabidopsis under the control of the 35S promoter even in the absence of Al (Durrett et al., 2007).
Paddy rice is usually cultivated under reduced soil conditions, where excess Fe2+ is present. Therefore, paddy rice has developed a strategy to prevent excess Fe uptake by oxidation of Fe2+ to insoluble Fe3+ on root surfaces (Fig. 3; Horiguchi, 1995 The concentration of citrate in the xylem sap was much higher than that of Fe, even in the knockout line (Fig. 5A). The question, therefore, arises why Fe deficiency-induced chlorosis in the knockout line occurred at low external Fe concentrations (Fig. 3A). This is probably due to the complexation of citrate with other cations. In the xylem sap, other cations, such as calcium (Ca), magnesium (Mg) are present at higher concentrations than Fe; therefore, most citrates form complexes with these cations. In fact, speciation simulation with GeoChem software showed that only a small percentage of total citrate in the xylem sap chelates Fe. In the OsFRDL1 knockout line (ND), the citrate concentration in the xylem sap did not decrease to zero (Fig. 5A). This suggests that there are other transporters either for citrate or for citrate-metal complexes. In the rice genome, there are five more homologs of OsFRDL1 (Fig. 1); therefore, the functions of these homologs should be examined in the future in terms of release of citrate to the xylem.
Overall, the function of OsFRDL1 is similar to AtFRD3 in terms of localization, transport, substrate, expression pattern, and phenotype of knockout lines. In Arabidopsis, knockout of AtFRD3 resulted in overaccumulation of Fe in the shoots (Rogers and Guerinot, 2002 In conclusion, our results clearly demonstrate that OsFRDL1 is a transporter of citrate, which is required for efficient translocation of Fe under limited Fe conditions. It is constitutively expressed and localized at the pericycle cells of rice roots.
Plant Materials and Growth Conditions Two rice (Oryza sativa) Tos-17 insertion lines, ND8025 and NC2637 for OsFRDL1, were obtained from the rice genome resource center. The homozygous lines were screened by PCR using OsFRDL1-specific primers (5'-GGTCTCACTGTTGTGCTTGGG-3' and 5'-GCTATGGTCCTCAGGCTCATG-3') and a left-border Tos-17 primer (5'-ATTGTTAGGTTGCAAGTTAGTTAAGA-3'). Progeny for genetic analysis was derived from a heterozygous line of ND8025 selected by PCR as described above. Seeds including wild-type rice (cv Nipponbare), two Tos-17 insertion homozygous lines, and the heterozygous progeny of ND8025 were soaked in water overnight at 25°C in the dark and then transferred to a net floating on 0.5 mM CaCl2 solution. On day 7, seedlings were transferred to a 3.5-L plastic pot containing one-half-strength Kimura B solution and grown in a greenhouse at 22°C to 25°C. After 10 d, the seedlings (five plants per pot) were transferred to a 1.2-L pot containing freshly prepared nutrient solution. The nutrient solution contained the macronutrients (mM): (NH4)2SO4 (0.18), MgSO4·7H2O (0.27), KNO3 (0.09), Ca(NO3)2·4H2O (0.18), and KH2PO4 (0.09), and the micronutrients (µM): MnCl2·4H2O (0.5), H3BO3 (3), (NH4)6Mo7O24·4H2O (1), ZnSO4·7H2O (0.4) and CuSO4·5H2O (0.2), supplied with either 0.2 or 10 µM FeSO4. The pH of this solution was adjusted to 5.5 and the nutrient solution was renewed every 2 d. Fe2+ was not oxidized during 2 d. After 10 to 15 d more growth, the seedlings were used for following experiments. Each experiment was repeated at least three times with three replicates each.
Plants cultivated with different Fe concentrations for 15 d as described above were harvested and separated into the roots and shoots. After drying at 70°C for 2 d, the samples were ground to fine powder and digested with 5 mL of 11 M HNO3 for 5 h at 150°C. The metal concentration was then determined by atomic absorption spectrometry (Z-2000; Hitachi).
Before collection of xylem sap, the nutrient solution was renewed. After 6 h, xylem sap was collected from the cut end for 1 h with a micropipette after decapitating the plant 3 cm above the roots. The concentration of Fe and other cations was determined using flameless atomic absorption spectrometry (Z-2000; Hitachi). Malate concentrations in the sap were analyzed with HPLC using an ion-exclusion column (Shimpack SCR-102H; 8.0 mm x 30 cm; Shimadzu) with a guard column (6.0 mm x 5 cm). Citrate concentrations in the xylem sap were analyzed with HPLC using a reverse-phase column (Cosmossil Packed Column 5C18-PAQ 4.6 i.d. x 250 mm; Nakalai tesque). The mobile phase was a dilute perchloric acid solution (pH 2.1) run at 40°C, and peaks were detected by postcolumn bromothymol blue method at the wavelength of 425 nm (Ma et al., 1997
Xylem sap collected as described above were immediately used for determination of Fe3+ and Fe2+ by QuantiChrom iron assay kit (Bioassay System) according to the manufacturer's instructions.
Perls blue staining was performed with the roots of wild-type rice, two Tos-17 lines, and a ND heterozygous progeny. Briefly, equal amounts of solutions of 4% (v/v) HCl and 4% (w/v) potassium ferrocyanide were mixed immediately prior to use. Seedlings (1-week-old) were exposed to the staining solution and vacuum infiltrated for 15 min. The seedlings were then rinsed with water and approximately 200-µm cross-sections were prepared by free hand. The staining was observed under optical microscope.
Total RNA was extracted from the roots and leaves of rice plants subjected to Fe deficiency for 1, 3, 6, and 8 d. The relative transcript levels of OsFRDL1, OsIRT1, and histone H3 (internal control) were determined by quantitative real-time RT-PCR as described previously (Yamaji and Ma, 2007
The synthetic peptide C-EEKTAAAAAAPEDLPA (positions 102–107 of OsFRDL1) was used to immunize rabbits to obtain antibodies against OsFRDL1. The roots of both wild-type and Tos-17 line (ND) grown at 10 µM FeSO4 were used for immunostaining as described previously (Yamaji and Ma, 2007
To compare organic acids secreted from rice roots in response to Al, root exudates from both Nipponbare and the knockout line (ND) were collected. Seedlings were exposed to a 0.5 mM CaCl2 (pH 4.5) solution overnight and then to a 0.5 mM CaCl2 (pH 4.5) solution containing 50 µM AlCl3 for 24 h. Root exudates collected were then passed through a cation-exchange resin column (16 x 14 mm) filled with 5 g of Amberlite IR-120B resin (H+ form), followed by an anion-exchange resin column (16 x 14 mm) filled with 2 g of AG 1 x 8 resin (100–200 mesh; formate form). Organic acids retained on an anion-exchange resin were eluted with 2 N HCl and the eluate was concentrated to dryness with a rotary evaporator (40°C). After evaporation, the residue was dissolved in 1 mL of milli-Q water; the concentration of organic acids was analyzed by enzymatic method according to Delhaize et al. (1993)
Oocytes were isolated from adult female Xenopus laevis frogs as described before (Ma et al., 2006
We thank the Rice Genome Resource Center for providing Tos-17 seeds. Received August 16, 2008; accepted November 10, 2008; published November 14, 2008.
1 This work was supported by the Program of Promotion of Basic Research Activities for Innovative Biosciences, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant no. 18380052 to J.F.M.), by a Sunbor grant, and by the Ohara Foundation for Agricultural Science.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jian Feng Ma (maj{at}rib.okayama-u.ac.jp).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128132 * Corresponding author; e-mail maj{at}rib.okayama-u.ac.jp.
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