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. 2012 Jan 3;33(2):117–126. doi: 10.1007/s10059-012-2165-9

Activation of Rice Yellow Stripe1-Like 16 (OsYSL16) Enhances Iron Efficiency

Sichul Lee 1, Nayeon Ryoo 2, Jong-Seong Jeon 2, Mary Lou Guerinot 1, Gynheung An 3,*
PMCID: PMC3887722  PMID: 22228180

Abstract

Graminaceous plants release ferric-chelating phytosiderophores that bind to iron. These ferric-phytosiderophore complexes are transported across the plasma membrane by a protein produced from Yellow Stripe 1 (YS1). Here, we report the characterization of OsYSL16, one of the YS1-like genes in rice. Real-time analysis revealed that this gene was constitutively expressed irrespective of metal status. Promoter fusions of OsYSL16 to β-glucuronidase (GUS) showed that OsYSL16 was highly expressed in the vascular tissues of the root, leaf, and spikelet, and in leaf mesophyll cells. The OsYSL16-green fluorescence protein (GFP) fusion protein was localized to the plasma membrane. From a pool of rice T-DNA insertional lines, we identified two independent activation-tagging mutants in OsYSL16. On an Fe-deficient medium, those mutants retained relatively high chlorophyll concentrations compared with the wild-type (WT) controls, indicating that they are more tolerant to a lack of iron. The Fe concentration in shoots was also higher in the OsYSL16 activation lines than in the WT. During germination, the rate of Fe-utilization from the seeds was higher in the OsYSL16 activation lines than in the WT seeds. Our results suggest that the function of OsYSL16 in Fe-homeostasis is to enable distribution of iron within a plant.

Keywords: activation tagging, homeostasis, iron

INTRODUCTION

Iron is an essential micronutrient with numerous cellular functions for all living organisms. Although Fe is abundant in the earth’s crust, it is often unavailable to plants because, at a neutral pH, it tends to form insoluble ferric oxide complexes in aerobic environments (Guerinot and Yi, 1994). Therefore, Fe-homeostasis in the whole organism, as well as in the cells, must be balanced to supply enough Fe for cell metabolism while avoiding excessive, toxic levels.

Plant cells actuate two separate mechanisms for acquiring Fe (Mori, 1999). Reduction-based Strategy I operates mainly in dicotyledonous and non-graminaceous plants, where Fe(III) is reduced to Fe(II) before being transported into the cell by an Fe(II) transporter. Robinson et al. (1999) have cloned Arabidopsis Fe(III)-chelate reductase (FRO2). After reduction to Fe(II), Fe is transported across the root plasma membrane via Fe(II) transporters (Marschner and Römheld, 1994). AtIRT1 is a major Fe-transporter at the root surface (Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002). AtIRT2, a close homolog, is located at the intracellular vesicles and, in cooperation with AtIRT1, maintains Fe-homeostasis in root epidermal cells (Vert et al., 2009).

Grasses, such as rice, corn and wheat, use the Chelation-based Strategy II. In response to Fe-deficiency, graminaceous plant cells release phytosiderophores (PSs), which belong to the mugineic acid (MA) family and are derived from the precursor nicotianamine (NA). Molecules bind to Fe(III) and specific plasma membrane transporter proteins import the Fe(III)-PS complexes (Römheld and Marschner, 1986). The molecular mechanism controlling Fe(III)-uptake has been elucidated by cloning the membrane transporter from the maize yellow stripe 1 (ys1) mutant, which shows characteristic interveinal chlorosis (Basso et al., 1994; Curie et al., 2001). Because that mutant is deficient in Fe(III)-PS uptake, it has been suggested that YS1 is the Fe(III)-PS transporter. The YS1 protein is up-regulated by Fe-deficiencies in roots and shoots, and functions as a proton-coupled symporter to transport Fe(III)-PS (Roberts et al., 2004; Schaaf et al., 2004). Yeast and oocyte studies have indicated that YS1 might have other substrates, including Zn, Ni, and Cu (Roberts et al., 2004; Schaaf et al., 2004).

Among the 18 YS1-like (OsYSL) genes in rice, OsYSL15 transports Fe(III)-deoxymugineic acid (DMA) and Fe(II)-NA (Inoue et al., 2009; Lee et al., 2009). OsYSL15 expression is strongly induced in the roots and shoots by Fe-deficiency. Under such conditions, osysl15 mutants exhibit chlorotic phenotypes and have reduced Fe concentrations in their shoots, roots, and seeds, thereby demonstrating roles for OsYSL15 in Fe-uptake and distribution in rice plants (Inoue et al., 2009; Lee et al., 2009). In barley, HvYS1 is a specific transporter for Fe(III)-PS, which is involved in primary Fe-acquisition in the roots (Murata et al., 2006). Other YSL members may play roles in long-distance movement of metals. OsYSL2, induced by Fe-deficiency, is localized to the plasma membrane and transports Fe(II)-NA and Mn(II)-NA, but not Fe(III)-DMA (Koike et al., 2004). An RNAi line of OsYSL2 shows decreased Fe-translocation to seeds, lower Fe concentrations in shoots and seeds, and greater accumulation of Fe in the roots (Ishimaru et al., 2010). Plants over-expressing OsYSL2 also have less Fe-translocation to shoots and seeds, indicating that OsYSL2 is a critical Fe(II)-NA transporter (Ishimaru et al., 2010). OsYSL18 is a transporter of Fe(III)-DMA but not of Fe(II)-NA (Aoyama et al., 2009). Its expression in flowers and the phloem of lamina joints implies that it is involved in translocating Fe in reproductive organs and phloem joints.

In Arabidopsis, YSL proteins are not secreted for Fe-uptake. Instead, they are proposed to function in transporting of Fe within a plant through the formation of Fe-NA complexes (Colangelo and Guerinot, 2006; Curie et al., 2001). AtYSL1 is a shoot-specific gene whose transcript level is increased in response to high-Fe conditions; its expression in young siliques suggests a role for AtYSL1 in Fe-loading to seeds (Le Jean et al., 2005). The ysl1/ysl3 double mutants exhibit Fe-deficiency symptoms, including interveinal chlorosis and decreased fertility (Waters et al., 2006). Concentrations of Fe, Zn, Cu, and Mn are altered in those double mutants, and seeds accumulate signifycantly less of all those metals except Mn. During leaf sensecence, both genes are strongly expressed throughout the leaves, implying that AtYSL1 and AtYSL3 function in moving metal-NA complexes during senescence and seed production. Chu et al. (2010) also have reported that AtYSL1 and AtYSL3 can transport Fe-NA, and that the latter transports Fe-DMA as well. The AtYSL2promoter::GUS fusion stains xylem-associated cells within the vasculature of expanded leaves. Its broad expression pattern within differentiated roots suggests that AtYSL2 participates in the lateral transport of metals in the veins (Di Donato et al., 2004; Schaaf et al., 2005). Finally, TcYSL3 encodes an NA-Ni/Fe-transporter in the metal hyper-accumulator Thlaspi caerulescens (Gendre et al., 2007).

In this study, we showed that the function of OsYSL16 in iron homeostasis is for distribution of Fe within rice plants.

MATERIALS AND METHODS

Plant growth

Wild-type (WT) and transgenic rice seeds were germinated on MS agar plates. The standard medium contained 0.1 μM CuSO4, 100 μM Fe(III)-EDTA, 30 μM ZnSO4, and 10 μM MnSO4 as micronutrients. To test the effect of deficiencies, we germinated seeds and then reared the seedlings on MS media lacking CuSO4 (Cu-deficient), Fe(III)-EDTA (Fe-deficient), ZnSO4 (Zn-deficient), or MnSO4 (Mn-deficient). For RNA analysis, shoot and root samples from 7-day-old seedlings were frozen with liquid nitrogen. Other seedlings were transplanted into soil and grown to maturity in a greenhouse (14-h photoperiod).

RNA isolation and mRNA quantification

Total RNA was isolated from frozen samples with RNAiso Plus (Takara, Japan). For cDNA synthesis, we used 2 μg of total RNA as template and M-MLV reverse transcriptase (Promega, USA) in a 25-μl reaction mixture. RT-PCR was conducted in a 50-μl solution containing a 1-μl aliquot of the cDNA reaction, 0.2 μM of gene-specific primers, 10 mM dNTPs, and 1 unit of rTaq DNA polymerase (Takara). The PCR products were separated by electrophoresis on a 1.2% agarose gel. Gene-specific primers for the OsYSL gene family are listed in Supplementary Table I. Real-time PCR was performed with a Rotor-Gene 6000 real-time rotary analyzer (Corbett Life Science, Australia) and a SYBR premix Ex Taq kit (Takara). Levels of actin1 mRNA were used to normalize the expression ratio for each gene. Changes in expression were calculated via the cycle threshold (ΔΔCt) method.

Genotyping OsYSL16 mutant plants

One insertional mutant line (1A13505) and two activation-tagging lines (1B04137 and 2D00022) for OsYSL16 were isolated from our T-DNA flanking sequence tag database (An et al., 2003; Jeong et al., 2006). For genotyping of the knockout mutant (osysl16-1), we used two gene-specific primers (F1, 5′-CTGGGGCTGAACAAGAAGAC-3′ and R1, 5′-GATGCCGAAATACCTGAGGA-3′) and a T-DNA-specific primer (LB, 5′-ACGTCCGCAATGTGTTATTAA-3′). For Line 1B04137 (OsYSL16-D1), two gene-specific primers (F2, 5′-ACATGAGGTTTGCATTTGCGTAC-3′ and R2, 5′-GAGACGACGATCAGACGAACG-3′) and a T-DNA-specific primer (LB, 5′-ACGTCCGCAATGTGTTATTAA-3′) were used. Line 2D00022 (OsYSL16-D2) required two gene-specific primers (F3, 5′-TGGGCTTCTAAAGTTTGGATT-3′ and R3, 5′-GCCAAAGTCCTTCACTTTCTC-3′) and a T-DNA-specific primer (RB, 5′-CAAGTTAGTCATGTAATTAGCCAC-3′). Levels of OsYSL16 transcript were determined by RT-PCR, using cDNA prepared from the leaves of 10-day-old seedlings.

Generation of OsYSL16 promoter::GUS fusion transgenic plants, and GUS assays

Genomic sequences (−1,700 bp to −1 bp from the translation initiation site) containing the promoter region of OsYSL16 were amplified by PCR, using two primers (pf, 5′-AAAAGCTTAATTAAGATCGCGTAGATTGAA-3′ and pr, 5′-AAGGATCCCTGCCGGCCGCACCCGACGAGGC-3′). This fragment was connected to the promoterless GUS gene (derived from pBI 101.2) and ligated into pCAMBIA1302, resulting in pGA2865. The new plasmid was transferred to Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method (An et al., 1988). Transgenic plants carrying the above construct were generated via Agrobacterium-mediated co-cultivation (Lee et al., 1999). Histochemical GUS-staining of the transgenic plants was performed according to the procedure described by Lee et al. (2009). Afterward, 10-μm sections were prepared and observed with a microscope (Nikon, Japan) under bright and dark-field illumination.

Sub-cellular localization of the OsYSL16-GFP fusion protein

The cDNA fragment spanning the entire ORF of OsYSL16 was amplified with primers Y16-pgf (5′-AAAGATCTCCGCCACGCGCTGGGCGGCGGT-3′; added BglII site is underlined) and Y16-pgr (5′-AAACTAGTGTTTCCCGGTATGAACTTCAT-3′; SpeI site underlined). After being digested with both BglII and SpeI, it was inserted into the BglII /SpeI-linearized pCAMBIA1302 vector. The resulting construct, pGA2886, containing the fusion of OsYSL16-GFP under the CaMV35S promoter, was confirmed by sequencing. The vector was then transferred to A. tumefaciens strain LBA4404 and introduced into rice as described above. Transgenic progeny plants were grown on sterile solid MS and collected at 5 days after germination. Their roots were examined by laser-scanning confocal microscopy (LSM 510 META, Carl Zeiss, Germany). GFP signals were detected by exciting at 488 nm from an argon laser and collecting emission at 522 nm.

Generation of OsYSL16-antisense transgenic plants

For our OsYSL16 antisense construction, its gene-specific sequence located at the 3′ end was amplified by primer pair anF (5′-AAGGTACCACAGCGACTGCTGTTCTCATAA-3′) and anR (5′-AAGAGCTCCGCCTTGTAGAAGAGGAAGAAG-3′). The PCR product was cut with KpnI and sacI, then ligated into the maize Ubiquitin1 (Ubi1) promoter of pGA1611, thereby generating pGA2876 (Lee et al., 1999). For RNA gel blot analysis, total RNAs were isolated from seedling leaves with an RNA isolation kit (Tri Reagent, USA). The isolated total RNAs were fractionated on a 1.3% agarose gel, blotted onto a nylon membrane, and hybridized with a 32P-labeled probe (Kang et al., 1998).

Measurements of plant growth and metal concentrations

Seeds of WT and mutant plants were germinated and seedlings grown on a solid medium containing MS salts with or without 100 μM Fe(III)-EDTA. For determining chlorophyll concentrations, the leaves were harvested and chlorophyll was extracted with 1 ml of 80% acetone from 0.1-g samples. After homogenization, the samples were incubated for 15 min and spun at 15,000 g for 10 min. An aliquot of supernatant fraction was then taken to measure the A663 and A643 with a spectrophotometer. Chlorophyll concentrations, including chlorophyll a and b, were obtained according to the method of Arnon (1949). For measuring levels of Fe, Zn, Cu, and Mn, the seeds, shoots, and roots were dried separately for 2 days at 70°C before being weighed. Samples were then digested in 1 ml of 11 N HNO3 for 3 days. After dilution, their metal concentrations were determined by atomic absorption spectroscopy (AAS; SpectraAA-800, USA).

RESULTS

Expression analysis of four OsYSL genes under different metal statuses

A database search for rice genome sequences identified a family of 18 genes that are similar to maize YS1 (Koike et al., 2004). Among them, OsYSL2, OsYSL9, OsYSL15 and OsYSL16 are grouped with YS1. Previously, we showed that, except for OsYSL16, transcripts of those genes are increased when Fe is limited (Lee et al., 2009). In this study, we studied expression patterns under various metal-deficient conditions.

Seedlings were grown for 7 days on an MS medium containing either standard amounts of micronutrients or else lacking Fe, Zn, Cu, or Mn. Afterward, gene expression was comprehensively examined by quantitative real-time PCR (Fig. 1). In shoots, transcript levels for OsYSL2 and OsYSL15 were very low when plants were grown under mineral-rich conditions, but were strongly induced by Fe-deficiency. Similar results were achieved when evaluating the roots. These observations suggest that both genes are needed when Fe is limiting. Likewise, OsYSL9 was induced by Fe-deficiency in the shoots but not the roots. By contrast, OsYSL16 was constitutively expressed in both roots and shoots at levels similar to those of the other three genes, but altering the Fe status had no remarkable effect on the expression of that gene. These observations coincide with those we previously reported (Lee et al., 2009). As for the other micronutrients (i.e., Cu, Zn, and Mn), their deficiencies elicited no such gene response under our experimental conditions (Fig. 1).

Fig. 1.

Fig. 1.

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Expression analyses of OsYSL genes under micronutrient-deficient conditions. Seedlings were grown for 7 d on MS medium containing standard amounts of micronutrients [0.1 μM CuSO4, 100 μM Fe(III)-EDTA, 30 μM ZnSO4, and 10 μM MnSO4]. For deficiency tests, seeds were germinated and seedlings grown for 7 d in MS medium lacking Fe(III)-EDTA (Fe-deficient), ZnSO4 (Zn-deficient), CuSO4 (Cu-deficient), or MnSO4 (Mn-deficient). Transcript levels are presented as ratios between mRNA levels of OsYSL2 (A), OsYSL9 (B), OsYSL15 (C), or OsYSL16 (D) and those of rice actin1. Vertical bars indicate standard deviation. Each value is average of 3 independent experiments.

We selected OsYSL16 for further study because it had not yet been characterized in detail.

Expression analysis of the OsYSL16 promoter in transgenic rice

To examine various spatial and temporal expression patterns, we fused the 1.7-kb fragment carrying the OsYSL16 promoter to GUS and generated transgenic plants that expressed this construct. Histochemical GUS-staining of 5-day-old seedlings showed that, consistent with our quantitative real-time PCR analysis, GUS activity was detected in seedling shoots and roots under normal growing conditions (Fig. 2A). For example, in the cross sections of seminal roots, strong activity was observed in the vascular cylinder (Fig. 2B). Within the differentiation zone, GUS expression was expanded to all regions except the epidermis (Fig. 2C). In lateral roots, activity was abundant in vascular bundle tissues (Fig. 2D) whereas the seedling shoots showed higher activity in younger leaves (Fig. 2E), where it was detected mainly in the vascular bundles and mesophyll cells but absent in the epidermis (Figs. 2F and 2G). In developing spikelets, GUS activity was constitutively detected at all developmental stages (Fig. 2H). Cross sections of flowers showed that activity was weak in the anther walls but detectable in the palea/lemma, particularly in the vascular bundle tissues (Fig. 2I). In developing seeds, all tissues had activity, including the embryo, seed coat, and endosperm (Figs. 2J and 2K).

Fig. 2.

Fig. 2.

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OsYSL16 promoter-driven GUS expression pattern. (A) Five-day-old seedling grown on MS medium. S, shoots; R, roots. (B–G), Cross sections of (B) 5-day-old seedling root at dividing zone (a in Fig. 2A), (C) differentiation zone (b in Fig. 2A), (D) lateral root from 10-day-old seedling, (E) shoot (c in Fig. 2A), (F) midrib region of leaf, and (G) leaf blade. (H) Temporal and spatial expression patterns of OsYSL16::GUS fusion construct in spikelets at various stages, as defined by extent of anther development. Samples; 1, tetrad; 2, early young microspore; 3, late young microspore; 4, vacuolated pollen; and 5, late pollen mitosis. (I) Cross section of Sample 5 in (H) at indicated position. (J) Analyses of GUS activity in developing seeds. Samples; 1, 3 DAP (days after pol-lination); 2, 5 DAP; 3, 7 DAP; 4, 10 DAP; 5, 20 DAP. (K) Cross section of Sample 4 in (J) at indicated position. (B), (E), (I), and (K) are dark-field images where GUS activities are visualized as red signals. In other panels, GUS-staining is shown in blue. A, anther; AL, aleurone layer; AR, aerenchyma; B, bulliform cell; C, cortex; E, endosperm; ED, endodermis; EP, epidermis; EX, exodermis; L, lemma; LR, lateral root; M mesophyll; P, palea; R, roots; S; shoots; SC, seed coat; V, vascular bundle. Bars = 1 cm in A; 50 μm in B-G; 1 mm in H, J; 100 μm in I, K.

OsYSL16 is localized in the plasma membrane

Because micronutrients are transported not only across the plasma membrane but also inside cellular compartments, we analyzed the cellular localization of OsYSL16. The PSORT program (http://psort.nibb.ac.jp) predicted, with high probability, that OsYSL16 was located in the plasma membrane. To confirm this prediction, we constructed a chimeric molecule encoding a fusion between OsYSL16 and GFP. In plants stably transformed with the construct, a GFP signal was found in the plasma membrane (Fig. 3). In contrast, fluorescence of GFP alone was observed in the cytosol and nuclei. Thus, we could conclude that this protein was responsible for metal transport in the plasma membrane.

Fig. 3.

Fig. 3.

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Sub-cellular localization of OsYSL16-GFP fusion protein. Full-length OsYSL16 cDNA was fused to GFP in same reading flame and resulting construct was transferred to Agrobacterium tumefaciens for rice transformation. Representative fluorescence microscopic images of roots expressing OsYSL16-GFP fusion protein are shown. GFP fluorescence was detected at plasma membrane. Roots expressing GFP under con-trol of CaMV35S promoter (P35S-GFP) were used as controls. GFP fluorescence in control was detected in cytosol and nuclei. Fluorescence (left) and bright-field (mi-ddle) images are overlaid at right.

Isolation of a OsYSL16 knockout mutant and generation of OsYSL16 antisense lines

Mutants are valuable tools for revealing the roles of a particular gene in physiological and developmental processes. From our rice flanking sequence-tag database (An et al., 2003; Jeong et al., 2006), we identified a mutant line, osysl16-1 (1A13505), in which T-DNA is inserted in the 2nd intron of OsYSL16 (Fig. 4A). T2 progeny were obtained and homozygous knockout plants were selected by PCR, using gene-specific primers and T-DNA primers. We demonstrated that OsYSL16 transcription was disrupted in osysl16-1 (Fig. 4B).

Fig. 4.

Fig. 4.

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Isolation of loss-of-function mutant in OsYSL16 and generation of antisense transgenic plants. (A) Schematic diagram of T-DNA insertion site in osysl16-1. Dark boxes show 7 exons; connecting black lines are 6 introns. T-DNA was inserted into 2nd intron. F1 and R1 are gene-specific primers, and LB is T-DNA-specific primer for genotyping. Fw and Rv are gene-specific primers for RT-PCR analysis. (B) RT-PCR analyses of OsYSL16 expression using OsYSL16-specific primers (Fw and Rv). Total RNA was prepared from osysl16-1 and WT seedlings. Rice Actin1 (OsAct1) mRNA was amplified to show equal loading of RNA in each sample. (C) Schematic diagram of pGA2876 expressing OsYSL16-antisense transcripts under control of maize Ubiquitin promoter (Pubi). anF and anR are gene-specific primers for generation of antisense construct. (D) RNA gel blot analysis of OsYSL16-antisense transgenic plants. A gene-specific region amplified by primers anF and anR was used as probe. Two WT and eight independent transgenic plants were examined. The rRNA levels are shown as controls (bottom). (E) and (F) Phenotypes of WT plants, osysl16-1, and OsYSL16-antisense transgenics (AN-1) germinated and grown on control MS (MS), Fe-deficient (Fe-), or Zn-deficient (Zn-) media for 10 days. Bars = 5 cm.

To evaluate the functional roles of OsYSL16 in Fe and Zn transport, we germinated seeds from WT and osysl16-1 plants and grew their seedlings on either a standard MS medium or media lacking Fe or Zn. Regardless of growing conditions, phenotypes were indistinguishable between mutants and WT plants, and heights and chlorophyll concentrations were not altered (Fig. 4E; Supplementary Figs. S1A and S1B). To assess whether the disruption of OsYSL16 affects distribution of Fe and Zn, we measured their levels in shoots and roots at various developmental stages. Concentrations of iron did not differ significantly between genotypes when the Fe supply was either sufficient or deficient (Supplementary Fig. S1C). For zinc as well, plants under either sufficient or deficient conditions showed no changes in concentrations regardless of genotype (Supplementary Fig. S1D).

Because OsYSL16 promoter activity was detected during flower and seed development (Figs. 2H and 2J), we postulated that disruption of that gene might affect Fe and Zn accumulations in the grains. However, concentrations of these minerals were similar between WT and osysl16-1 seeds (Supplementary Figs. S1E and S1F).

To confirm our results with the OsYSL16 loss-of-function mutant, we generated eight independent transgenic plants expressing OsYSL16 in an antisense orientation under the control of the maize Ubi1 promoter (Fig. 4C). Their antisense transcript levels were determined by Northern blot analysis using RNA samples prepared from transgenic leaves (Fig. 4D). From the six lines that strongly expressed the antisense transcript, we selected Lines #1 (AN-1) and #6 (AN-6) for further analysis. No remarkable differences in plant heights or chlorophyll concentrations were found between the WT and antisense transgenic plants grown in the absence or presence of Fe or Zn (Fig. 4F; Supplementary Figs. S1A and S1B). Moreover, antisense suppression of OsYSL16 did not affect the Fe and Zn concentrations in seeds of AN-1 and AN-6 (Supplementary Figs. S1E and S1F).

We also analyzed Cu and Mn distributions in WT and osysl16 mutant plants. Under deficiencies of either, no phenotypic alterations were apparent (data not shown). Levels of Cu and Mn were also unchanged in the shoots, roots, and mature seeds (data not shown). Therefore, all of these results indicate that another gene(s) must compensate for the deficiency of OsYSL16 in response to some metals.

Isolation of activation-tagging mutants of OsYSL16

Because our loss-of-function approaches did not provide any useful information toward understanding the roles of OsYSL16, we isolated activation-tagging mutants to study gain-of-function. In Line 1B04137 (OsYSL16-D1), the 35S 4× enhancer elements were inserted approximately 0.9 kb upstream from the OsYSL16 coding region (Fig. 5A). For Line 2D00022 (OsYSL16-D2), those elements were about 6.5 kb downstream of OsYSL16 (Fig. 5A). In these lines, OsYSL16 was a reasonable candidate for the activation-tagging gene. T2 progeny were obtained and homozygous plants were selected by PCR using gene-specific primers and a T-DNA primer (Fig. 5A). Subsequent quantitative real-time PCR analysis revealed that, compared with the control, transcript levels of OsYSL16-D1 and OsYSL16-D2 were increased by 7.7- and 2.9-fold in the leaves, and by 7.0- and 3.2-fold in the roots, respectively (Fig. 5B).

Fig. 5.

Fig. 5.

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Phenotypes of OsYSL16 activation-tagging mutants. (A) Schematic representation of T-DNA insertion sites in OsYSL16-D1 and OsYSL16-D2 plants. Primers (F2, F3, R2, R3, LB, and RB) used for genotyping are shown by horizontal arrows. 4 × 35S denotes 4 copies of 35S enhancer element. (B) Quantitative real-time PCR analyses of WT and mutant seedlings using gene-specific primers (Fw and Rv) and RNA samples from shoots (S) and roots (R). Transcript levels are presented as ratios between mRNA levels of OsYSL16 and those of rice actin1. D1 and D2 indicate OsYSL16-D1 and OsYSL-D2 plants; WT means segregating WT plants. Vertical bars indicate standard deviation. Each value is average of 3 independent experiments. (C) Phenotypes of OsYSL16-D1 at seedling stage under Fe- and Zn-deficiencies. Lower panels are enlargements of second leaves. Pictures were taken 10 d after germination. Bar = 5 cm. (D) Total chlorophyll concentrations from WT plants and OsYSL16-D1 mutants grown under Fe-sufficient (MS) or -deficient (Fe-) conditions for 4 to 10 DAG. Data are from 2 independent experiments. In each, 4 plants were used for measuring chlorophyll. Error bars represent SE. Significant differences from WT were determined by Student’s t-test. *, P < 0.05.

Characterization of OsYSL16 activation-tagging mutants

Homozygous activation-tagging plants of OsYSL16-D1 and OsYSL16-D2, and their co-segregating WT progeny, were grown on either a standard MS medium or one lacking Fe or Zn. Heights and chlorophyll concentrations did not differ between the mutants and the WT for plants on the standard medium or those grown under a Zn-deficiency (Figs. 5C and 5D; Supplementary Figs. S2A and S2C). Likewise, when Cu or Mn was lacking, the WT plants were indistinguishable from the activation-tagging mutants (data not shown).

However, when Fe was deficient, the homozygous activation plants were eventually greener than the control (Figs. 5C and 5D; Supplementary Figs. S2A and S2C). For example, at 4 DAG, no differences were found between WT and activation-tagging plants when Fe was lacking in the medium. By 7 DAG, leaf chlorosis had appeared and chlorophyll concentrations were reduced in plants grown under Fe-limiting conditions. However, the chlorophyll concentration was about 10% higher in the mutants. At 10 DAG, the activation-tagging plants had accumulated at least 20% more chlorophyll compared with the WT, indicating that the OsYSL16-D plants were more tolerant of an Fe-deficiency.

Activation of OsYSL16 affects Fe distribution

We evaluated Fe and Zn concentrations for 10 days in plants grown on either standard or deficient media. For the standard MS medium, Fe levels in shoots and roots were similarly increased for both mutant and WT plants (Fig. 6A). In Fe-deficient media, Fe concentrations did not differ between genotypes at 4 DAG (Fig. 6B). However, at 7 and 10 DAG, the OsYSL16-D1 plants contained more Fe in the shoots but less in the roots compared with the segregating WT control (Fig. 6C). These results suggested that activation of OsYSL16 causes enhanced translocation of Fe from roots to shoots.

Fig. 6.

Fig. 6.

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Phenotypic analyses of WT and OsYSL16-D1 plants. (A, B), Fe concentrations in shoots and roots of WT and mutants grown for 10 days in Fe-sufficient (MS) or Fe-deficient (Fe-) medium. Fe (C) and Zn (D) concentrations in mature seeds. Fe (E) concentrations from mature seeds and from seeds of 4–10 DAG seedlings grown under Fe-deficient conditions. Error bars represent SE. Significant differences from WT were determined by Student’s t-test. *, P < 0.05.

Over time, Zn concentrations remain relatively constant in shoots but decreased in roots (Supplementary Fig. S3A). In the Zn-deficient medium, OsYSL-D1 plants contained the same amount of Zn as from the WT (Supplementary Fig. S3B). Levels of Cu and Mn were also unchanged in the shoots and roots of either type (data not shown). Therefore, OsYSL16 does not appear to play a role in transporting Zn, Cu, or Mn in rice plants.

Because OsYSL16 is actively expressed during seed development (Fig. 2J), we measured mineral levels in mature grains to elucidate how enhanced expression might influence micronutrient storage. Here, Fe, Zn, Cu, and Mn concentrations were similar among the WT and two activation-tagging plants (Figs. 6C and 6D). Moreover, individual seed weights were not significantly different between transgenics and the WT (data not shown). This indicated that the increased Fe concentration in OsYSL16-D1 shoots was not due to a higher amount of stored Fe in mature seeds. Therefore, it was more likely that Fe-translocation to the shoots was more efficient in the mutants. To verify this hypothesis, we measured Fe concentrations in seeds from plants grown on an Fe-deficient medium. As expected, the level of Fe was more rapidly decreased in OsYSL16-D1 seeds compared with the segregating WT controls (Fig. 6E). However, Zn levels did not differ between OsYSL16-D1 and the WT (Supplementary Fig. S3C). These results supported our supposition that OsYSL16 functions to mobilize Fe preferentially to the shoots.

DISCUSSION

In this study of rice, we demonstrated that enhanced tolerance to a low-Fe environment can be achieved through overexpression of OsYSL16. This gene is one of four that are closely related to maize YS, which encodes an Fe(III)-PS transporter (Koike et al., 2004). Here, expression of OsYSL2, OsYSL9, and OsYSL15 was induced by Fe-deficiency whereas transcript levels of OsYSL16 were relatively constant under either sufficient or deficient conditions (Fig. 1). Both OsYSL2 and OsYSL15 are very close together on Chromosome 4 while OsYSL9 and OsYSL16 are closely located on Chromosome 2. The protein sequences of OsYSL2 and OsYSL15 are 72% identical to each other; those of OsYSL9 and OsYSL16 are 74% identical. These findings indicate that gene duplication occurred during recent genome evolution and that functioning is redundant.

In fact, a knockout mutation and antisense suppression of OsYSL16 did not cause any alteration in Fe distribution and utilization (Fig. 4; Supplementary Fig. S1). This was likely due to functional redundancy. OsYSL16 and OsYSL9 are highly homologous, closely located, and show a similar induction pattern in roots by external metal supply. These suggest that OsYLS9 could complenent the loss of OsYSL16. To test this hypothesis, generation of the double knock-out of these two genes will be needed. Insertion mutagenesis usually generates recessive loss-of-function mutations, making them unsuitable for functional analysis of redundant genes. Because many rice genes are members of a multigene family (Goff et al., 2002; Yu et al., 2002), researchers must utilize alternative tools for their investigation. Activation-tagging is one method for evaluating the function of redundant genes because the effect is dominant. From a pool of rice T-DNA insertion lines, we obtained two independent activation-tagging lines with enhanced expression of OsYSL16. To analyze the phenotypes of these mutants, we tested their tolerance to Fe-deficiency by growing the plants on a medium lacking iron. Visual differences between OsYSL16-D and the WT were also reflected in their chlorophyll concentrations and, therefore, the former were deemed more tolerant of that deficiency. Under those conditions, the Fe concentration was higher in the shoots of OsYSL16-activated mutants than in the WT plants, and the mutants also had a more rapid rate of Fe-mobilization from seeds to shoots. Ferritin is the major iron stroage protein in all living orgisms (Harrison and Arosio, 1996). In the shoots of OsYSL16-activiated mutants, transcript levels of rice ferriitn genes were higher than those of WT (data not shown), corresponding with the increased level of Fe in the transgenic shoots. However, OsYSL16-D plants did not show any phenotypic differences under deficiencies of Zn, Cu, or Mn (Figs. 5 and 6; Supplementary Figs. S2 and S3). Therefore, these results suggest that OsYSL16 functions to distribute Fe preferentially within a plant.

Ectopic expression of OsYSL16 also recovered the Fe-deficiency (data not shown). However, the extent of that rescue was weaker compared with expression in the activation-tagging lines. Such ectopic expression alters the original pattern when a foreign promoter is used. In contrast, activation-tagging generally upholds the characteristics of the target genes (Jeong et al., 2006). Therefore, it appears that maintaining a proper expression pattern is a more efficient way to achieve the enhanced effects conferred by a gene.

Previoulsy, we showed that overexpression of two rice Fe transpoters, OsIRT1 and OsYSL15, resulted in shorter heights and fewer tiller than WT when grown in the paddy field (Lee and An, 2009; Lee et al., 2009). However, transgenics from OsYSL16-activated mutants and overexpressors did not show any alteration in plant architecture.

Although OsYSL16 is expressed in many cell types, it is more preferentially expressed in the vascular tissues of roots and leaves. This pattern is similar to that of AtYSL2, which is expressed most strongly in the vascular tissues of leaves and primary roots (Di Donato et al., 2004; Schaff et al., 2005). AtYSL1 is expressed in the xylem parenchyma of leaves, where it is up-regulated in response to excess Fe, as well as in pollen and young siliques (Le Jean et al., 2005). In rice, OsYSL2 is expressed in the companion cells and phloem of leaves (Koike et al., 2004). Transcripts of TcYSL3 and TcYSL7 are localized in the central cylinder of young roots, the phloem of older roots, and, in the case of TcYSL7, in the phloem of the stem (Gendre et al., 2007). However, OsYSL16 is also expressed in the mesophyll, suggesting its role for Fe-utilization in many cell types.

Arabidopsis IRT1 and rice OsIRT1 and OsYSL15 are expressed strongly in the outer tissues of the roots, including root hairs and epidermis, especially under Fe-deficiency, thereby playing a role in iron-uptake from the soil (Ishimaru et al., 2006; Lee et al., 2009; Vert et al., 2002). However, we did not see such expression by OsYSL16 in the root epidermal cells. Furthermore, enhanced expression of that gene could not increase Fe levels in the vegetative tissues and mature seeds from that found under normal growing conditions. Therefore, all of our results allow us to postulate that the primary function of OsYSL16 for Fe-homeostasis is in distributing iron within a plant.

Supplementary Material

Acknowledgments

We thank Kyungsook An and In-Soon Park for generating the transgenic lines, and Priscilla Licht for English editing of the manuscript. This work was supported in part by grants from the Next-Generation BioGreen 21 Program (No. PJ008215), Rural Development Administration, Republic of Korea; the Basic Research Promotion Fund, Republic of Korea (KRF-2007-341-C00028); Kyung Hee University (20110269); and the U.S. National Science Foundation Plant Genome Program (DBI-0701119 to M.L.G.).

Note:

Supplementary information is available on the Molecules and Cells website (www.molcells.org).

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