Plant Mol Biol (2015) 88:165–176 DOI 10.1007/s11103-015-0315-0

Iron deficiency regulated OsOPT7 is essential for iron homeostasis in rice Khurram Bashir1,2 · Yasuhiro Ishimaru1,3 · Reiko Nakanishi Itai1 · Takeshi Senoura4 · Michiko Takahashi1 · Gynheung An5 · Takaya Oikawa3 · Minoru Ueda3 · Aiko Sato6 · Nobuyuki Uozumi6 · Hiromi Nakanishi1 · Naoko K. Nishizawa1,4 

Received: 1 December 2014 / Accepted: 1 April 2015 / Published online: 18 April 2015 © Springer Science+Business Media Dordrecht 2015

Abstract  The molecular mechanism of iron (Fe) uptake and transport in plants are well-characterized; however, many components of Fe homeostasis remain unclear. We cloned iron-deficiency-regulated oligopeptide transporter 7 (OsOPT7) from rice. OsOPT7 localized to the plasma membrane and did not transport Fe(III)-DMA or Fe(II)-NA and GSH in Xenopus laevis oocytes. Furthermore OsOPT7 did not complement the growth of yeast fet3fet4 mutant. OsOPT7 was specifically upregulated in response to Fedeficiency. Promoter GUS analysis revealed that OsOPT7

expresses in root tips, root vascular tissue and shoots as well as during seed development. Microarray analysis of OsOPT7 knockout 1 (opt7–1) revealed the upregulation of Fe-deficiency-responsive genes in plants grown under Fesufficient conditions, despite the high Fe and ferritin concentrations in shoot tissue indicating that Fe may not be available for physiological functions. Plants overexpressing OsOPT7 do not exhibit any phenotype and do not accumulate more Fe compared to wild type plants. These results indicate that OsOPT7 may be involved in Fe transport in rice.

Khurram Bashir and Yasuhiro Ishimaru have contributed equally to this work.

Keywords  Oryza sativa · Iron · Iron deficiency · Ferritin · Oligopeptide transporter

Electronic supplementary material  The online version of this article (doi:10.1007/s11103-015-0315-0) contains supplementary material, which is available to authorized users. * Naoko K. Nishizawa [email protected]‑tokyo.ac.jp 1

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1‑1‑1 Yayoi, Bunkyo‑ku, Tokyo 113‑8657, Japan

2

Center for Sustainable Resource Science, RIKEN, Yokohama Campus, 1‑7‑22 Suehiro‑cho, Tsurumi‑ku, Yokohama, Kanagawa 230‑0045, Japan

3

Graduate School of Science, Tohoku University, 6‑3, Aramaki‑aza Aoba, Aoba‑ku, Sendai 980‑8578, Japan

4

Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1‑308 Suematsu, Nonoichi‑shi, Ishikawa 921‑8836, Japan

5

Department of Plant Molecular Systems, Biotech and Crop Biotech Institute, Kyung Hee University, Yongin 446‑701, Republic of Korea

6

Graduate School of Engineering, Tohoku University, 6‑6‑07, Aobayama, Aoba‑ku, Sendai, Japan











Abbreviations OPT Oligopeptide transporter Fe Iron

Introduction Iron (Fe) is an essential micronutrient for living organisms, and Fe-deficiency is a serious nutritional problem that affects ~30 % of the global population (World Health Organization 2002; Bashir et al. 2013c; Lingam et al. 2011). In plants, Fe is required for several cellular processes, such as respiration, chlorophyll biosynthesis, and photosynthetic electron transport, and plant growth is significantly impaired under Fe-deficient conditions (Guerinot and Ying 1994). Although Fe is abundant in soils, it is not readily available since it is present mainly as oxidized compounds that are poorly soluble in neutral to alkaline soils (Marschner 1995). Plants have developed sophisticated mechanisms to acquire Fe from soil (Marschner et al. 1986;

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Mori 1999). Graminaceous plants synthesize and secrete ferric Fe-chelating compounds known as mugineic acid family phytosiderophores (MAs) from their roots through transporter of mugineic acid 1 (TOM1) to solubilize Fe (Takagi 1976; Takagi et al. 1984; Nozoye et al. 2011). The resulting Fe-MA complexes are then reabsorbed into the roots through the yellow stripe 1 (YS1) family transporters (Curie et al. 2001; Inoue et al. 2009; Lee et al. 2009). MAs are synthesized from methionine (L-Met). Nicotianamine synthase (NAS) catalyzes the formation of nicotianamine (NA), which is then converted into a 3′-keto intermediate, and the subsequent reduction of the 3′-carbon of the keto intermediate produces deoxymugineic acid (DMA). DMA is the first MA synthesized in the pathway. The genes involved in MA biosynthesis have been characterized, and most of these genes are regulated by the Fe status of plants (Bashir et al. 2006; Bashir and Nishizawa 2006; Lingam et al. 2011; Kobayashi and Nishizawa 2012; Bashir et al. 2010). After acquisition from soil, Fe is transported to leaves and developing seeds through a complex and complicated molecular mechanisms (Bashir et al. 2013a; Ishimaru et al. 2011b; Bashir et al. 2011b; Yokosho et al. 2009; Yamaji and Ma 2014). Moreover, cellular distribution of Fe is also very important and disturbance in cellular Fe homeostasis results in significant alteration in cellular metabolism (Bashir et al. 2011a, c, 2013b; Kim et al. 2006; Nozoye et al. 2014a, b; Zhang et al. 2012). YSL family oligopeptide transporters play a significant role in distribution of Fe to shoots and fluorescence. OsYSL2 transports Fe(II)-NA and Mn(II)-NA (Koike et al. 2004), and is important for the translocation of Fe and manganese (Mn) via the phloem (Ishimaru et al. 2010) while OsYSL16 plays a role in the allocation of Fe(III)-deoxymugineic acid via the vascular bundles (Kakei et al. 2012). Excess Fe produces highly toxic hydroxyl radicals, and to avoid cellular damage, plants must store Fe carefully. Plants primarily store Fe bound to ferritin, a multimeric protein that can bind up to 4500 Fe atoms, and which plays an important role in Fe homeostasis (Harrison and Arosio 1996). The expression of ferritin increases with Fe availability (to avoid cellular damage), and decreases when Fe is deficient (Briat et al. 2010). The ferritin genes from a number of plant species have been cloned. Ferritin localizes mainly to plastids, and its regulation in response to Fe and other environmental stresses has been investigated extensively (Briat et al. 2010). In this report, we cloned and characterized rice iron deficiency regulated oligopeptide transporter 7 (OsOPT7). OsOPT7 belongs to the oligopeptide transporter (OPT) family, which has members in archaebacteria, bacteria, fungi, and plants, but not in animals (Ryu et al. 2009; Stacey et al. 2008). OsOPT7-knockout mutant (opt7–1) accumulated more Fe than wild type (WT) plants and in such

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plants expression of the Fe-deficiency responsive genes in shoot tissue was triggered, indicative of Fe unavailability. The substrate for OsOPT7 has not been determined; however present data indicate its role in Fe distribution within plant especially in shoot tissue. Understanding the regulation of OsOPT7 by Fe availability will facilitate the development of strategies to mitigate this serious agricultural and nutritional problem.

Materials and methods Cloning of OsOPT7 Iron-deficiency-regulated oligopeptide transporter 7 (OsOPT7) was identified as a putative member of the OPT family upregulated under Fe- deficient conditions through microarray analysis. The full-length rice cDNA clone for OsOPT7 (AK102404) was acquired from the Rice Fulllength cDNA Database (KOME; http://cdna01.dna.affrc. go.jp/cDNA/). The orthologs of OsOPT7 were identified through BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). GSH transport assay A 2274-bp fragment of OsOPT7 produced by digestion with EcoRI and XbaI was inserted into the HindIII and EcoRI sites of the pYES vector. The resulting plasmid, pYESOsOPT7, was linearized by NotI digestion. Capped complementary RNA (cRNA) was synthesized in vitro using the MEGAscript SP6 kit (Ambion, Austin, TX, USA). For GSH uptake experiments oocytes of Xenopus laevis frogs were injected with either capped complementary RNA encoding OsOPT7 or water and incubated at 17 °C for 48 h (Kato et al. 2001). Then oocytes were placed in Barth-MES; pH 5, 6 or 7) containing 100 µM GSH, (20 nM 3H-labeled GSH) After 10 h incubation, oocytes were washed with 100 µl Barth-MES solution, followed by the addition of 100 µl of 10 % SDS, vortexed briefly and radioactivity of the oocytes was measured in a liquid scintillation analyzer as described previously (Ishimaru et al. 2011b; Bashir et al. 2011b). For NA and DMA transport experiments, OsOPT7 was cloned into pGEM-3zf(+) vector and substrate transport activity for NA and DMA was measured in oocytes at −80 and −100 mV, as described previously (Koike et al. 2004). For yeast complementation assay, full length ORF of OsOPT7 was cloned into the expression vector pDR195 and then introduced into yeast cells using the lithium acetate method. Saccharomyces cerevisiae strains used were wild type parental strain (BY4741; MATalpha, his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), and ferrous Fe uptake-defective double mutant Δfet3fet4 (MATalpha, his3Δ1; leu2Δ0; met15Δ0;

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ura3Δ0; YMR058w::kanMX4; YMR319c::kanMX4). OsNRAMP5 which transports cadmium, Mn and Fe was used as positive control. For media without Fe, 20 mM bathophenanthroline disulfonic acid (BPDS) was added. After spotting at three dilutions (optical densities at 600 nm of 0.1, 0.01, and 0.001), the plates were incubated at 30 °C for 4 days.

microarray (Agilent Technologies, CA), as described previously (Ishimaru et al. 2009). A signal value of Fe-deficiency-treated plants >500, a P value 2 in both the Cy3 and Cy5 channels was considered indicative of significant upregulation.

Rice transformation and growth conditions

Samples for Fe-deficiency and Fe excess were prepared as described previously (Bashir et al. 2014). For RT analysis primers for OsOPT7 were forward 5′-CGTCATCATCACCATCTTCG-3′and reverse 5′- GCCCTGAAGAGTGAGACCTG-3′ respectively. Forward and reverse primers for α-tubulin were 5′-TCTTCCACCCTGAGCAGCTC-3′ and 5′-AACCTTGGAGACCAGTGCAG-3′ respectively. Northern blot analyses were performed using the full-length ORF of OsOPT7 according to the method described previously (Englerblum et al. 1993; Yoshihara et al. 2003; Inoue et al. 2009). Western blot analyses were performed with 10 μg of total purified protein from opt7–1 and WT plants using antibodies raised against ferritin soybean (Goto et al. 1998).

The 1.8-kb 5′-flanking region of the OsOPT7 gene was amplified by PCR using genomic DNA as template with the forward primer 5′- GAGAAAGCTTTGGCACCACCACCTGCATGCCTCAG-3′ and the reverse primer 5′-GAGAAAGCTTCTCCCTAGCCTCGATCTCCTTCCTC-3′, which contain a HindIII restriction site. The amplified fragment was fused into the pBluescript II SK+ vector, and its sequence was confirmed. The OsOPT7 promoter was digested with XhoI and BglII, and the digested 1.8-kb fragment was subcloned upstream of the uidA ORF, which encodes β-glucuronidase, in the pIG121Hm vector. Oryza sativa L. cv. Tsukinohikari was transformed with this vector, plants were grown in a greenhouse, and histochemical localization of OsOPT7 was evaluated in three independent T2 plants, as described previously (Bashir et al. 2011c; Ishimaru et al. 2006). To generate OsOPT7-OX plants, OsOPT7 was subcloned into pIG121Hm under the control of the CaMV35 S promoter. Oligo‑DNA microarray analysis The OsOPT7 T-DNA knockout line (opt7–1) and opt7–2 were obtained from rice functional genomics database maintained at http://signal.salk.edu/cgi-bin/RiceGE. Isolation of the opt7–1 homozygous mutant was performed using PCR based screening using a T-DNA right border specific primer 5′-AATATCTGCATCGGCGAACTGATCG-3′ and OsOPT7 specific primers 5′-GCGTTGAAGTTGAAATTGAAAGAAAACC-3′ and 5′-CAGCAAGCCAAGCATGACGTCACTTAG-3′. Seeds of rice (O. sativa L. cv. Hwayoung) were germinated on wet filter paper and cultured as described previously (Bashir et al. 2007). For Fedeficiency treatments, 17 days old plants were transferred to culture solution lacking Fe. Roots and leaves were harvested after 7 days, frozen in liquid nitrogen, and stored at −80 °C until use. RNA was extracted from the roots and shoots of three plants, and total RNA samples (200 ng) from opt7–1 and WT plants were labeled with Cy3 or Cy5 using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, CA). Microarray analysis was performed in duplicate according to the manufacturer’s instructions using the rice 44 K oligo-DNA

Real time PCR, northern and western blot analyses

Determination of metal concentrations The elemental analysis of the WT and opt7–1-knockout plants were performed using inductively coupled plasma atomic emission spectrometry (SPS1200VR; Seiko, Tokyo, Japan), as described previously (Ishimaru et al. 2007, 2009, 2011a; Bashir et al. 2013b). Chloroplasts were isolated from hydroponically grown WT and opt7-1 plants with chloroplast isolation kit (Sigma Aldrich) according to the manufacturer’s instructions, broken using the freeze–thaw method, and chlorophyll content was measured using acetone as described previously (Porra et al. 1989). Metal concentration was measured as described previously (Ishimaru et al. 2009; Bashir et al. 2011c). Subcellular localization of OsOPT7 The full-length ORF of OsOPT7 was amplified using the forward and reverse primers 5′-CACCATGGCGTCGTTGAAGTCGCCGGTG-3′ and 5′-TCAGAACGGGCATCCCTTGAC-3′, respectively, and was subcloned into pENTR/D-TOPO (Invitrogen) and sequenced using a Thermo Sequenase cycle sequencing kit (Shimadzu, Kyoto, Japan) and a DNA sequencer (DSQ-2000L; Shimadzu). The OsOPT7 ORF was then subcloned into pH7WGF2 (Karimi et al. 2002) using the LR recombination reaction (Invitrogen). Onion epidermal cells were transformed and expression of GFP was assessed as described previously (Ishimaru et al. 2009).

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b

0.03

a

Copies per α-tubulin

a Copies per α-tubulin

Fig. 1  Expression and subcellular localization of OsOPT7. a–b ▸ Real Time PCR analysis in response to Fe-deficiency and Fe excess in roots (a) and shoots (b). c–g Subcellular localization of OsOPT7 in onion epidermal cells. c Fluorescence of sGFP only, d. OsOPT7sGFP fluorescence, e transverse image, f fluorescence of FM4-64 and j overlay of FM4-64 and OsOPT7-sGFP. OsOPT7 was transiently expressed in onion epidermal cells and observed using confocal microscopy. h–j. GSH transport assay in xenopus leavis oocytes a at pH5, h at pH 6 i and at pH7 j. k-l. Serial dilutions of yeast cells for WT transformed with empty vector (V.C.) and Δfet3fet4 (Fe uptake mutant) transformed with V.C., OsOPT7 or OsNRAMP5 and grown on Fe sufficient SD medium (k) and Fe deficient medium supplemented with 20 µM BPDS. Values followed by different letters are significantly different according to analysis of variance followed by the Student–Newman–Keuls test (n = 3; a, p