Rice OsYSL15 Is an Iron-regulated Iron(III)-Deoxymugineic Acid Transporter Expressed in the Roots and Is Essential for Iron Uptake in Early Growth of the Seedlings
2008; Elsevier BV; Volume: 284; Issue: 6 Linguagem: Inglês
10.1074/jbc.m806042200
ISSN1083-351X
AutoresHaruhiko Inoue, Takanori Kobayashi, Tomoko Nozoye, Michiko Takahashi, Yusuke Kakei, Kazumasa Suzuki, Mikio Nakazono, Hiromi Nakanishi, Satoshi Mori, Naoko K. Nishizawa,
Tópico(s)Soil Carbon and Nitrogen Dynamics
ResumoGraminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-β-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth. Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-β-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth. Iron is essential for virtually all living organisms. Iron deficiency is the most widespread human nutritional problem in the world. There are two billion anemic people worldwide, and ∼50% of all anemia cases can be attributed to iron deficiency (1Mason J.B. Lotfi M. Dalmiya N. Sethuraman K. Deitchler M. Geibel S. Gillenwater K. Gilman A. Mason K. Mock N. The Micronutrient Report: Current Progress and Trends in the Control of Vitamin A, Iodine, and Iron Deficiencies. The Micronutrient Initiative/International Development Research Center, Ottawa, Canada2001: 1-40Google Scholar). In plants, iron plays a key role in electron transfer in both photosynthetic and respiratory reactions in chloroplasts and mitochondria. Although abundant in mineral soils, iron is sparingly soluble under aerobic conditions at high soil pH. Consequently, plants grown on calcareous soils often exhibit severe chlorosis because of iron deficiency, which is a major agricultural problem resulting in reduced crop yields (2Marschner H. Mineral Nutrition of Higher Plants.2nd Ed. Academic Press, London, UK1995: 313-324Crossref Google Scholar). Higher plants have two strategies for the uptake of oxidized Fe(III) from the rhizosphere (3Römheld V. Marschner H. Plant Physiol.. 1986; 80: 175-180Google Scholar). All higher plants except graminaceous plants take up iron by using ferric-chelate reductases to reduce ferric iron to Fe(II), which is absorbed by ferrous iron transporters (strategy I (4Eide D. Broderuis M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A.. 1996; 93: 5624-5628Google Scholar, 5Robinson N.J. Procter C.M. Connolly E.L. Guerinot M.L. Nature.. 1999; 397: 694-697Google Scholar, 6Vert G. Grotz N. Dedaldechamp F. Gaymard F. Guerinot M.L. Briat J.F. Curie C. Plant Cell.. 2002; 14: 1223-1233Google Scholar)). Alternatively, graminaceous plants secrete iron chelators called mugineic acid family phytosiderophores (MAs) 4The abbreviations used are: MA, mugineic acid family phytosiderophore; DMA, 2′-deoxymugineic acid; GFP, green fluorescent protein; GUS, β-glucuronidase; VC, vector control; MS, Murashige and Skoog medium; RT, reverse transcription; ORF, open reading frame; MES, 4-morpholineethanesulfonic acid; NA, nicotianamine; TM, transmembrane domain; IDEF, iron deficiency-responsive element-binding factor; CBS, circadian clock-associated 1-binding site. 4The abbreviations used are: MA, mugineic acid family phytosiderophore; DMA, 2′-deoxymugineic acid; GFP, green fluorescent protein; GUS, β-glucuronidase; VC, vector control; MS, Murashige and Skoog medium; RT, reverse transcription; ORF, open reading frame; MES, 4-morpholineethanesulfonic acid; NA, nicotianamine; TM, transmembrane domain; IDEF, iron deficiency-responsive element-binding factor; CBS, circadian clock-associated 1-binding site. from their roots to solubilize iron in the rhizosphere (strategy II (3Römheld V. 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The genes that encode the biosynthetic enzymes in the reaction pathway converting S-adenosylmethionine to MAs have been isolated and characterized in barley (HvNAS1–7, NASHOR1 and -2, HvNAAT-A and -B, and HvDMAS1 (16Higuchi K. Suzuki K. Nakanishi H. Yamaguchi H. Nishizawa N.K. Mori S. Plant Physiol.. 1999; 119: 471-479Google Scholar, 17Herbik A. Koch G. Mock H.P. Dushkov M. Czihal A. Thielmann J. Stephan U.W. Bäumlein H. Eur. J. Biochem.. 1999; 265: 231-239Google Scholar, 18Takahashi M. Yamaguchi H. Nakanishi H. Shioiri T. Nishizawa N.K. Mori S. Plant Physiol.. 1999; 121: 947-956Google Scholar, 19Bashir K. Inoue H. Nagasaka S. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. J. Biol. Chem.. 2006; 281: 32395-32402Google Scholar)) and rice (OsNAS1–3, OsNAAT1, and OsDMAS1 (19Bashir K. Inoue H. Nagasaka S. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. J. Biol. Chem.. 2006; 281: 32395-32402Google Scholar, 20Higuchi K. Watanabe S. Takahashi M. Kawasaki S. Nakanishi H. Nishizawa N.K. Mori S. Plant J.. 2001; 25: 159-167Google Scholar, 21Inoue H. Takahashi M. Kobayashi T. Suzuki M. Nakanishi H. Mori S. Nishizawa N.K. Plant Mol. Biol.. 2008; 66: 193-203Google Scholar)). Expression of these genes is strongly induced in response to iron deficiency. In rice, histochemical analysis of promoter-β-glucuronidase (GUS) transformants revealed that OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1 share highly similar expression patterns, with significant expression in all cells of iron-deficient roots especially in the companion and pericycle cells (19Bashir K. Inoue H. Nagasaka S. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. J. Biol. Chem.. 2006; 281: 32395-32402Google Scholar, 21Inoue H. Takahashi M. Kobayashi T. Suzuki M. Nakanishi H. Mori S. Nishizawa N.K. Plant Mol. Biol.. 2008; 66: 193-203Google Scholar, 22Inoue H. Higuchi K. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2003; 36: 366-381Google Scholar), where DMA is thought to be synthesized. Cloning of the maize YS1 (yellow stripe 1) gene (10Curie C. Panavience Z. Loulergue C. Dellaporta S.L. Briat J.F. Walker E.L. Nature.. 2001; 409: 346-349Google Scholar) led to the identification of the specific transporters responsible for uptake of iron-chelated MAs complexes from the rhizosphere into root cells. The maize ys1 mutant is defective in Fe(III)-MAs uptake (23von Wirén N. Mori S. Marschner H. Römheld V. Plant Physiol.. 1994; 106: 71-77Google Scholar). YS1 expression is increased in both roots and shoots under conditions of iron deficiency, but it is not strongly affected by zinc or copper deficiency (10Curie C. Panavience Z. Loulergue C. Dellaporta S.L. Briat J.F. Walker E.L. Nature.. 2001; 409: 346-349Google Scholar, 24Roberts L.A. Pierson A.J. Panaviene Z. Walker E.L. Plant Physiol.. 2004; 135: 112-120Google Scholar). Schaaf et al. (25Schaaf G. Ludewig U. Erenoglu B.E. Mori S. Kitahara T. von Wirén N. J. Biol. Chem.. 2004; 279: 9091-9096Google Scholar) investigated the transport properties of YS1 in Xenopus oocytes by electrophysiological analysis. YS1 functions as a proton-coupled symporter for various DMA-bound metals including Fe(III), Zn(II), Cu(II), and Ni(II). YS1 also transports nicotianamine (NA)-chelated Ni(II), Fe(II), and Fe(III) complexes. Recently, a barley homolog of YS1 (HvYS1) has been identified (26Murata Y. Ma J.F. Yamaji N. Ueno D. Nomoto K. Iwashita T. Plant J.. 2006; 46: 563-572Google Scholar). In contrast to YS1, HvYS1 is highly specific for Fe(III)-MAs while demonstrating a low transport activity for MAs chelated to Zn(II), Cu(II), Ni(II), or Co(II). Non-graminaceous plants also possess YS1-like (YSL) genes that encode transporters considered to play important roles in internal metal homeostasis by transporting metal-NA complexes, as non-graminaceous plants synthesize NA but not MAs (27DiDonato Jr., R.J. Roberts L.A. Sanderson T. Eisley R.B. Walker E.L. Plant J.. 2004; 39: 403-414Google Scholar, 28Le Jean M. Schikora A. Mari S. Briat J.F. Curie C. Plant J.. 2005; 44: 769-782Google Scholar, 29Schaaf G. Schikora A. Haberle J. Vert G. Ludewig U. Briat J.F. Curie C. von Wirén N. Plant Cell Physiol.. 2005; 46: 762-774Google Scholar, 30Gendre D. Czernic P. Conéjéro G. Pianelli K. Briat J.F. Lebrun M. Mari S. Plant J.. 2006; 49: 1-15Google Scholar, 31Waters B.M. Chu H.S. DiDonato Jr., R.J. Roberts L.A. Eisley R.B. Lahner B. Salt D. Walker E.L. Plant Physiol.. 2006; 141: 1446-1458Google Scholar). Our previous search for YS1 homologs in the rice genome data base identified 18 putative OsYSL genes (32Koike S. Inoue H. Mizuno D. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2004; 39: 415-424Google Scholar). Among these are OsYSL2 transports Fe(II)-NA and Mn(II)-NA but not Fe(III)-MAs (32Koike S. Inoue H. Mizuno D. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2004; 39: 415-424Google Scholar). OsYSL2 expression is strongly induced in iron-deficient leaves with particularly strong expression in phloem cells of the leaves and leaf sheaths. These results suggest that OsYSL2 functions as an Fe(II)-NA transporter responsible for the phloem transport of iron (32Koike S. Inoue H. Mizuno D. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2004; 39: 415-424Google Scholar). To date, no rice OsYSL with a transport activity for Fe(III)-MAs has been identified. Here we describe that OsYSL15 encodes a functional Fe(III)-DMA transporter, whose expression pattern strongly indicates its involvement in Fe(III)-DMA uptake from the rhizosphere and phloem transport of iron. An essential role of OsYSL15 for transport of iron during early seedling growth is also suggested by the results presented here. Plant Materials—Nontransgenic and transgenic rice seeds were germinated on Murashige and Skoog (MS) medium and transferred to nutrient solution (11Mori S. Nishizawa N. Plant Cell Physiol.. 1987; 28: 1081-1092Google Scholar, 22Inoue H. Higuchi K. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2003; 36: 366-381Google Scholar) in a greenhouse with 30 °C light/25 °C dark periods under natural light conditions. The pH of the culture solution was adjusted daily to 5.3 with 1 m HCl. For micronutrient deficiency treatments, 3-week-old plants were transferred to nutrient solution without iron, zinc, manganese, or copper and grown for 3 more weeks. Time course expression analysis was carried out as described previously (33Nozoye T. Itai R.N. Nagasaka S. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Soil Sci. Plant Nutr.. 2004; 50: 1125-1131Google Scholar). Plants were grown under iron-deficient conditions for 2 weeks and were harvested at 3-h intervals after the lights were turned on (0 h). The lights were turned off at 14 h and were turned on again at 24 h. Flowers and seeds were obtained from iron-sufficient rice plants. Laser Microdissection and Expression Analysis—Laser microdissection was used to examine tissue-specific expression (34Nakazono M. Qiu F. Borsuk L. Schnable P.S. Plant Cell.. 2003; 15: 583-596Google Scholar). Iron-sufficient and iron-deficient roots were fixed by a 10-min infiltration of 3:1 ethanol:acetic acid into the tissues under vacuum on ice. The vials containing the samples in the fixative were then gently mixed on a rotator at 4 °C for 1 h. This fixation step was repeated twice with a fresh solution of fixative. The fixed samples were then transferred to 10% sucrose solution, and the tissues were infiltrated with this solution under vacuum for 10 min on ice, followed by gentle mixing on a rotator at 4 °C for 1 h. After replacement with a fresh sucrose solution, the vials were gently mixed overnight on a rotator at 4 °C. The fixed samples were then embedded in diethyl pyrocarbonate water, frozen in a dry ice hexane bath, and stored at –80 °C. The tissues were sectioned into 20-μm thick slices in a cryostat (Leica CM1850, Leica Microsystems) and mounted on polyethylene foil pretreated with a tissue-adhesive solution of 0.1% poly-l-lysine (Sigma) at –30 °C. The foil was attached to a glass slide and dried at room temperature. The sections were laser-microdissected with a Leica AS LMD (Leica Microsystems). Target tissues were selected and dissected from the sections (Fig. 1, a–d). Twenty sections of the epidermis/exodermis, cortex, and stele were dissected and separately collected in 0.5-ml sample tubes. To avoid contamination, we confirmed the identity of the tissues in each tube at every step. Total RNA was extracted from the microdissected samples using an RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer's instructions. The amount of total RNA was measured fluorometrically using a RiboGreen RNA quantitation kit (Molecular Probes, Eugene, OR) with 485 nm excitation and 530 nm emission wavelengths according to the manufacturer's instructions. Total RNA (15 ng) was used to synthesize first-strand cDNA using oligo(dT) primer and avian myeloblastosis virus reverse transcriptase XL (Takara, Japan). One-twentieth of the resulting cDNA sample was used in a 25-μl PCR with the specific primers shown in supplemental Table S1. As a control, rice actin-specific primers were used. The PCR products were analyzed by electrophoresis in agarose gels. Yeast Complementation—The following strains of Saccharomyces cerevisiae were used in this study: CM3260 (parent strain) MATα trp1-63 leu2-3, 112 gcn4-101 his3-609 ura3-52, and DEY1453 (fet3 fet4 mutant) MATα/MATα ade2/+ can1/can1 his3/his3 leu2/leu2 trp1/trp1 ura3/ura3 fet3-2::His3/fet3–2::HIS3 fet4-1::LEU2/fet4-1::LEU2. Yeast cells were grown in YPD (1% yeast extract, 2% peptone and 2% glucose) or SD medium supplied with the appropriate amino acids. Agar was added to 2% for solid plate media. Fe(III)-DMA complexes were prepared as described previously (25Schaaf G. Ludewig U. Erenoglu B.E. Mori S. Kitahara T. von Wirén N. J. Biol. Chem.. 2004; 279: 9091-9096Google Scholar); Fe(III)-DMA was prepared by mixing appropriate amounts of a 10 mm FeCl3 solution, pH < 2, and MES/Tris buffer (pH 7.5) and 100 mm DMA for 2 h at room temperature. The chelate solution was filtered through an Amicon ultrafree MC 0.22-μm filter unit (Millipore) to remove precipitated iron. The yeast expression vectors pDR195 and pDR195-YS1 (29Schaaf G. Schikora A. Haberle J. Vert G. Ludewig U. Briat J.F. Curie C. von Wirén N. Plant Cell Physiol.. 2005; 46: 762-774Google Scholar) were the kind gifts of Dr. Nicolaus von Wirén (University of Hohenheim, Germany). Restriction sites (XbaI, BamHI, and XhoI) were generated in the multicloning site using the following oligonucleotide linkers: 5′-GTCGACTCTAGAGGATCCCTCGAGTGAAGATCT-3′ and 5′-AGATCTTCACTCGAGGGATCCTCTAGAGTCGAC-3′. For expression of OsYSL15 in yeast, the subcloned OsYSL15 cDNA was excised using XbaI and XhoI sites and inserted into the same sites of the vector to form pDR195-OsYSL15. Yeast cells were transformed using the lithium acetate transformation method (35Gietz R.D. Schiestl R.H. Methods Mol. Cell. Biol.. 1995; 5: 255-269Google Scholar). For complementation assays, single colonies of transformed yeast cells were cultured in liquid SD medium without iron twice for 24 h. The cells were washed in 1 ml of 10 mm Tris, 500 mm EDTA (pH 7.5) twice before each incubation period. The A600 of the cultures was adjusted to 1.0, and 10 μl of 10× serial dilutions were spotted onto SD plates to test for complementation. Transport Activity of OsYSL15 in Xenopus laevis Oocytes—Electrophysiological measurement of OsYSL15 transport activity using X. laevis oocytes was carried out according to Ref. 32Koike S. Inoue H. Mizuno D. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2004; 39: 415-424Google Scholar. A 2019-bp fragment of OsYSL15 full-length cDNA was amplified by PCR using a cDNA pool prepared from iron-deficient rice roots and primers 5′-TCGTGGGAATTCTCGAGCAGCTAAGCGAGATCGACGC-3′ and 5′-TTTATTTCTAGAATCCTCCACCCATGAAATTAAACAC-3′. Six independent oocytes injected with OsYSL15 were used to measure currents. Substrate-induced currents were also measured in six independent water-injected oocytes as controls. Transport activity was measured by Hitachi, Ltd., Life Science Group (Japan). Northern Blot Analysis—RNA extraction and Northern blot analysis was performed as described previously (22Inoue H. Higuchi K. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2003; 36: 366-381Google Scholar). A probe specific for OsYSL15 was amplified by PCR using a cDNA pool prepared from iron-deficient rice roots and the same primers as those used for the RT-PCR analysis of microdissected tissues. Quantitative RT-PCR Analysis—Quantitative RT-PCR analysis of roots and seedlings was carried out as described previously (21Inoue H. Takahashi M. Kobayashi T. Suzuki M. Nakanishi H. Mori S. Nishizawa N.K. Plant Mol. Biol.. 2008; 66: 193-203Google Scholar) using the specific primers shown in supplemental Table S1. Subcellular Localization of OsYSL15-GFP—The open reading frame (ORF) of OsYSL15 was amplified using two primers: 5′-CACCATGGAGCACGCCGACGCGGACCGCA-3′ and 5′-GCTTCCAGGCGTAAACTTCATGCAG-3′. The amplified fragment was subcloned into pENTR/D-TOPO (Invitrogen). This entry vector was designated pENTR-OsYSL15. Using pDEST35S-sGFP plasmid (36Ishimaru Y. Suzuki M. Kobayashi T. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. J. Exp. Bot.. 2005; 56: 3207-3214Google Scholar) as the destination vector, LR recombination reactions (Invitrogen) between the destination vector and the entry vector generated an expression clone containing the gene encoding 35S-OsYSL15-sGFP. Onion epidermal cells were transformed using the Biolistic PDS-1000/He particle delivery system (Bio-Rad), and the transiently expressed sGFP fluorescence was observed using a laser-scanning confocal microscope (LSM510, Karl Zeiss) according to Ref. 37Mizuno D. Higuchi K. Sakamoto T. Nakanishi H. Mori S. Nishizawa N.K. Plant Physiol.. 2003; 132: 1989-1997Google Scholar. OsYSL15 Promoter-GUS Analysis—The 1.5-kb 5′-upstream region of the OsYSL15 gene (–1500 to –1 bp from the putative translation initiation codon) was amplified by PCR using genomic DNA as a template. The primers used were the forward primer 5′-GAGAGAAAGCTTGTATAGCATTTGACTCCGCGGACTT-3′ and the reverse primer 5′-GAGAGATCTAGAGGCGGCGGCGGCGGCGTCGATCTCG-3′. The amplified and verified fragment was excised using HindIII and XbaI and subcloned upstream of the uidA ORF, which encodes GUS, in the pIG121Hm vector (38Hiei Y. Ohta S. Komari T. Kumashiro T. Plant J.. 1994; 6: 271-282Google Scholar). An Agrobacterium tumefaciens strain (C58) carrying the above construct was used to transform rice (O. sativa L. cv. Tsukinohikari) as described previously (20Higuchi K. Watanabe S. Takahashi M. Kawasaki S. Nakanishi H. Nishizawa N.K. Mori S. Plant J.. 2001; 25: 159-167Google Scholar). T1 seeds were germinated and cultured as described above and subjected to GUS expression analysis as described previously (22Inoue H. Higuchi K. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2003; 36: 366-381Google Scholar). Histochemical staining during seed germination was carried out as described previously (39Nozoye T. Inoue H. Takahashi M. Ishimaru Y. Nakanishi H. Mori S. Nishizawa N.K. Plant Mol. Biol.. 2007; 64: 35-47Google Scholar). Generation and Characterization of OsYSL15 Knockdown Rice—To suppress OsYSL15 expression, a 209-bp fragment of the OsYSL15 gene was amplified by PCR with the primers OsYSL15iF 5′-CACCTGGAAGCTAAGAGGTGTAGTGTGTT-3′ and OsYSL15iR 5′-ATGCCAAACTAAACAATTCTCAAGT-3′, and the amplified fragment was cloned into a Gateway pENTR/D-TOPO cloning vector (Invitrogen). The verified fragment was transferred into pIG121-RNAi-DEST (40Ogo Y. Itai R.N. Nakanishi H. Kobayashi T. Takahashi M. Mori S. Nishizawa N.K. Plant J.. 2007; 51: 366-377Google Scholar) by an LR clonase reaction (Invitrogen), and used for rice transformation. Transgenic OsYSL15 knockdown (OsYSL15i) seeds were germinated on standard MS medium, iron-free MS medium, or MS medium with 10% (v/v) of Tetsuriki-aqua (containing ∼5 mm Fe(II)-citrate; Aichi Steel, Aichi, Japan (4141. Sasamoto, H., Yasui, M., and Mori, S. (2005) Abstracts, Plant Nutrition for Food Security, Human Health and Environmental Protection, pp. 1116–1117, Beijing, ChinaGoogle Scholar)). For quantitative RT-PCR analysis of OsYSL15, seeds were first germinated on MS medium with 10% (v/v) of Tetsuriki-aqua for 6 days and then transplanted to iron-free MS medium for 6 days. Laser Microdissection Expression Analysis of OsYSLs in Root Tissues—We previously used TBLASTN to search the rice (O. sativa L. ssp. Japonica cv. Nipponbare) genomic data base, resulting in 18 potential OsYSL genes (32Koike S. Inoue H. Mizuno D. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2004; 39: 415-424Google Scholar). To estimate possible functions of the 18 OsYSL genes, we performed RT-PCR in the epidermis/exodermis, cortex, and stele of iron-sufficient and -deficient roots (Fig. 1). Tissues were isolated using laser microdissection (34Nakazono M. Qiu F. Borsuk L. Schnable P.S. Plant Cell.. 2003; 15: 583-596Google Scholar) (Fig. 1, a–d). Expression of OsYSL15 and OsYSL16 was induced in the epidermis/exodermis and stele of iron-deficient roots (Fig. 1e). Expression of OsYSL5, OsYSL6, OsYSL7, OsYSL14, and OsYSL17 was detected in all tissues of both iron-sufficient and -deficient roots. Expression of OsYSL8 was down-regulated by iron deficiency, and expression of OsYSL12 was detected in the cortex and stele under both iron-sufficient and -deficient conditions. Expression of OsYSL13 was detected in both iron-sufficient and -deficient cortex. No expression of OsYSL1, OsYSL2, OsYSL3, OsYSL4, OsYSL9, OsYSL10, OsYSL11, and OsYSL18 was detected in any of the tissues analyzed. We chose to focus on the OsYSL15 gene because its expression was strongly up-regulated in the root epidermis under conditions of iron deficiency, indicating that its encoded protein is likely to play a role in iron uptake from the rhizosphere. Sequence and Protein Structure of OsYSL15—The OsYSL15 cDNA has a 2019-bp open reading frame (ORF). The gene is located on one arm of chromosome 2 and has seven exons, as do YS1 and OsYSL2 (10Curie C. Panavience Z. Loulergue C. Dellaporta S.L. Briat J.F. Walker E.L. Nature.. 2001; 409: 346-349Google Scholar, 32Koike S. Inoue H. Mizuno D. Takahashi M. Nakanishi H. Mori S. Nishizawa N.K. Plant J.. 2004; 39: 415-424Google Scholar). Notably, the length of each exon of OsYSL15 is comparable with the exons of OsYSL2. OsYSL15 and OsYSL2 are tandemly located on the genome and separated by a 26-kb interval (Fig. 2a). Based on the nucleotide sequence, OsYSL15 is predicted to encode a protein of 672 amino acids (Fig. 2b). OsYSL15 was most similar to the maize YS1 transporter (10Curie C. Panavience Z. Loulergue C. Dellaporta S.L. Briat J.F. Walker E.L. Nature.. 2001; 409: 346-349Google Scholar) compared with the other OsYSLs (76% amino acid identity; supplemental Fig. S1). Topological analysis of OsYSL15 using HMMTOP predicts 16 transmembrane domains (TMs) with the N terminus located in the cytoplasm. Despite the high degree of similarity overall between OsYSL15 and YS1, two regions had relatively low similarity to each other. The first region, between the N terminus and TM1, showed 42% identity (23/54 residues). In YS1, this region contains 48% of the glutamic acid residues of the protein (11/23 residues) (10Curie C. Panavience Z. Loulergue C. Dellaporta S.L. Briat J.F. Walker E.L. Nature.. 2001; 409: 346-349Google Scholar), although the corresponding region in OsYSL15 contains 39% (7/18 residues). The second region of relatively low similarity, located between TM8 and TM9, showed 53% identity between OsYSL15 and YS1 (25/47 residues). This region is predicted to constitute the longest loop sequence extending into the cytoplasm, which contains the region responsible for structural property and substrate specificity of YS1/YSL transporters (42Harada E. Sugase K. Namba K. Iwashita T. Murata Y. FEBS Lett.. 2007; 581: 4298-4302Google Scholar). Amino acid alignment of this region revealed moderate similarity between YS1 and OsYSL15 (12/20 residues) with a perfect match in eight sequential residues at the center (Fig. 2b, boxed region). OsYSL15 Transports Fe(III)-DMA Complexes—A yeast complementation assay using a fet3 fet4 mutant defective in high and low affinity iron uptake (43Dix D.R. Bridgham J.T. Broderius M.A. Byersdorfer C.A. Eide D.J. J. Biol. Chem.. 1994; 269: 26092-26099Google Scholar) was used to identify the transport activity of OsYSL15. OsYSL15 rescued the growth defect of the fet3 fet4 mutant on SD medium containing Fe(III)-DMA complexes (Fig. 3a). Maize YS1 also rescued the yeast strain from the growth defect, but the control vector did not, consistent with previous reports (10Curie C. Panavience Z. Loulergue C. Dellaporta S.L. Briat J.F. Walker E.L. Nature.. 2001; 409: 346-349Google Scholar, 25Schaaf G. Ludewig U. Erenoglu B.E. Mori S. Kitahara T. von Wirén N. J. Biol. Chem.. 2004; 279: 9091-9096Google Scholar). Introduction of OsYSL15 or YS1 had no effect on yeast grown on normal or iron-minus SD medium without Fe(III)-DMA (Fig. 3, b and c). Electrophysiological studies with X. laevis oocytes were used to further confirm substrates transported by OsYSL15. OsYSL15 was heterologously expressed in the oocytes, and the substrate-induced inward currents at –80 mV were measured in response to Fe(III)-DMA or several related compounds (Fig. 3d). OsYSL15 transported Fe(III)-DMA but did not transport Fe(II)-NA, Fe(III)-NA, or Mn(II)-NA complexes. Consistent results were observed under different clamping voltages (data not shown). Regulation of OsYSL15 Expression by Micronutrient Deficiencies and Daily Fluctuations—The upstream sequence of OsYSL15 was searched for known cis-acting elements. Among the elements related to iron deficiency response in rice plants (44Kobayashi T. Nakayama Y. Itai R.N. Nakanishi H. Yoshihara T. Mori S. Nishizawa N.K. Plant J.. 200
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