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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Disruption of OsYSL15 Leads to Iron Inefficiency in Rice Plants^{1,[C],[W],[OA]}

Department of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang 790–784, Republic of Korea (S.L., Y.L., G.A.); Department of Biology, University of Massachusetts, Amherst, Massachusetts 01003 (J.C.C., E.L.W.); and Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755 (S.A.K., M.L.G.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Uptake and translocation of metal nutrients are essential processes for plant growth. Graminaceous species release phytosiderophores that bind to Fe³⁺; these complexes are then transported across the plasma membrane. We have characterized OsYSL15, one of the rice (Oryza sativa) YS1-like (YSL) genes that are strongly induced by iron (Fe) deficiency. The OsYSL15 promoter fusion to β-glucuronidase showed that it was expressed in all root tissues when Fe was limited. In low-Fe leaves, the promoter became active in all tissues except epidermal cells. This activity was also detected in flowers and seeds. The OsYSL15:green fluorescent protein fusion was localized to the plasma membrane. OsYSL15 functionally complemented yeast strains defective in Fe uptake on media containing Fe³⁺-deoxymugineic acid and Fe²⁺-nicotianamine. Two insertional osysl15 mutants exhibited chlorotic phenotypes under Fe deficiency and had reduced Fe concentrations in their shoots, roots, and seeds. Nitric oxide treatment reversed this chlorosis under Fe-limiting conditions. Overexpression of OsYSL15 increased the Fe concentration in leaves and seeds from transgenic plants. Altogether, these results demonstrate roles for OsYSL15 in Fe uptake and distribution in rice plants.

Despite its abundance in soils, Fe is present as oxihydrates with low bioavailability. To avoid a deficiency, two distinct strategies are possible for Fe acquisition (Marschner et al., 1986). In strategy I, used by dicotyledonous and nongraminaceous monocotyledonous plants, Fe²⁺ transport is coupled to an Fe³⁺ chelate reduction step. Under Fe deficiency, protons are released via H⁺-ATP pumps into the rhizosphere to lower the soil pH, and subsequently, Fe³⁺-chelate reductase is induced to reduce Fe³⁺ to the more soluble Fe²⁺, which is then absorbed by a specific transporter. The plasmalemma root ferric-chelate reductase, FRO2, reduces soil Fe³⁺ (Robinson et al., 1999) and provides Fe²⁺ for IRT1, a major metal transporter that takes up Fe²⁺ into the root epidermis. This is evidenced by the lethal chlorotic phenotypes of IRT1 knockout mutants (Eide et al., 1996; Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002).

With strategy II, low Fe availability in the soil is overcome in grasses such as maize (Zea mays) and rice (Oryza sativa). In response to Fe deficiency, these crops synthesize and release nonproteinogenic amino acids in the mugineic acid family of phytosiderophores (MAs) to fix Fe³⁺ in the soil (Takagi et al., 1984). All MAs are synthesized from L-Met, sharing the same pathway from L-Met to 2'-deoxymugineic acid (DMA; Bashir et al., 2006). L-Met is adenosylated by S-adenosylmethionine synthetase (Takizawa et al., 1996). Nicotianamine synthase (NAS) catalyzes the trimerization of S-adenosylmethionine molecules to form nicotianamine (NA; Higuchi et al., 1999), which is then converted into a 3''-keto intermediate via the transfer of an amino group by nicotianamine aminotransferase (NAAT; Inoue et al., 2008). Genes encoding NAS and NAAT for MA biosynthesis have been isolated (Higuchi et al., 1999; Takahashi et al., 1999; Inoue et al., 2003, 2008). The subsequent reduction of the 3''-carbon in the keto intermediate produces DMA by DMA synthase (Bashir et al., 2006). In response to Fe deficiency, these grasses increase their production and secretion of MAs into the rhizosphere, where they chelate various metals, including Fe³⁺. The Fe³⁺-MA complexes are then taken up by an Fe³⁺-MA transporter. The Yellow Stripe1 (YS1) gene has been isolated and characterized in maize, where it encodes the high-affinity Fe³⁺-MA transporter (Curie et al., 2001). YS1 functions as a proton-coupled symporter for phytosiderophore (PS)-chelated metals (Curie et al., 2001; Roberts et al., 2004; Schaaf et al., 2004).

Among the 18 YS1-like (YSL) genes in rice, OsYSL2 is up-regulated by Fe deficiency in the leaves, particularly in the phloem, and is also expressed in developing seeds (Koike et al., 2004). OsYSL2:GFP is localized to the plasma membrane. Electrophysiological measurements using Xenopus laevis oocytes have shown that OsYSL2 transports Fe²⁺-NA and Mn²⁺-NA but not Fe³⁺-DMA (Koike et al., 2004). OsYSL15 encodes a functional Fe³⁺-DMA transporter whose expression pattern strongly indicates its involvement in Fe³⁺-DMA uptake from the rhizosphere and in phloem transport of Fe (Inoue et al., 2009). OsYSL15 knockdown seedlings are severely arrested in their germination and early growth but are rescued by a high Fe supply, demonstrating that OsYSL15 plays a crucial role in Fe homeostasis during the first stages of growth (Inoue et al., 2009). It is generally believed that most plants depend largely on a single strategy (I or II) for Fe acquisition. In graminaceous plants, such as barley (Hordeum vulgare) and maize, the inducible Fe²⁺ transporter system either is absent or is expressed at very low levels (Zaharieva and Romheld, 2001). Rice and its wild relatives are well adapted for growth under submerged conditions, in which Fe²⁺ is more abundant than Fe³⁺. Isolation of OsIRT1 and OsIRT2, which encode functional Fe²⁺ transporters (Bughio et al., 2002; Ishimaru et al., 2006), suggests that rice plants can take up Fe²⁺ even though they do not have inducible Fe²⁺ chelate reductase activity (Ishimaru et al., 2006). Analysis with the positron-emitting tracer imaging system has confirmed that, in addition to taking up Fe³⁺-DMA, rice plants can absorb Fe²⁺ from the root environment (Ishimaru et al., 2006).

The presence of YSL genes is not restricted to strategy II plants. In fact, eight orthologs (AtYSL1–AtYSL8) have been found in Arabidopsis (Curie et al., 2001), a genus that neither synthesizes nor uses MAs. NA is the biosynthetic precursor to MAs and is also a strong chelator of various transition metals (von Wirén et al., 1999). NA is ubiquitous in the plant kingdom, and these YSL proteins mediate the internal transport of metals bound to NA (Walker, 2002). AtYSL1 is a shoot-specific gene whose transcript levels increase in response to high-Fe conditions (Le Jean et al., 2005). It is also expressed in young siliques, suggesting a role in the Fe loading of seeds. Indeed, mutations in that gene reduce seed Fe content and delay germination, but those can be rescued by exogenous Fe. AtYSL2 transcript levels are decreased under Fe-deficient conditions; they also respond to copper (Cu) and zinc (Zn; DiDonato et al., 2004; Schaaf et al., 2005). Whereas single mutants of ysl1 and ysl3 have no visible phenotypes, the ysl1/ysl3 double mutants exhibit Fe deficiency symptoms. Likewise, their mobilization of Zn and Cu from the leaves is impaired during senescence, suggesting that the physiological roles for YSL1 and YSL3 are their delivery of metal micronutrients to and from vascular tissues (Waters et al., 2006). In the heavy-metal hyperaccumulator Thlaspi caerulescens, TcYSL3, a member of the YSL gene family, encodes a Ni/Fe-NA transporter (Gendre et al., 2007).

Nitric oxide (NO) is a bioactive molecule implicated in a number of physiological functions, including the intracellular and intercellular mediation of some animal responses (Anbar, 1995). It also serves as a cellular messenger in controlling growth, development, and adaptive responses to multiple stresses in higher plants (Durner and Klessig, 1999; Beligni and Lamattina, 2001; Neill et al., 2002; Lamattina et al., 2003; Graziano and Lamattina, 2005; Lopez-Bucio et al., 2006). NO stimulates the accumulation of both ferritin mRNA and protein, suggesting that it is a signaling molecule for modulating Fe homeostasis in plants (Murgia et al., 2002). NO probably acts through the regulation of a trans-acting factor, because its effect relies on the Fe-dependent regulatory sequence present on the Atfer1 promoter. NO appears to play a role in improving Fe availability within a plant, as demonstrated by an Fe-deficiency phenotype that is recovered by NO without changing the internal levels of total Fe (Graziano et al., 2002). In tomato (Solanum lycopersicum), the NO level is increased in deprived roots to improve Fe availability by controlling the expression of uptake-related genes and by regulating the physiological and morphological adaptive responses to Fe-deficient conditions (Graziano and Lamattina, 2007).

Here, we have determined the physiological roles of OsYSL15 in Fe homeostasis using two independent T-DNA insertional mutants and overexpression lines.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Expression Analysis of OsYSL Genes under Different Fe Concentrations

Rice has 18 putative OsYSL genes (Koike et al., 2004). Among them, OsYSL2, OsYSL9, OsYSL15, and OsYSL16 are phylogenetically grouped with maize YS1 (Koike et al., 2004). We used quantitative real-time PCR to investigate whether expression of these four rice genes is responsive to changes in the external Fe supply (Fig. 1 ). Seedlings were grown for 7 d on Murashige and Skoog (MS) medium with 0, 1, 10, 100, or 500 µM Fe. Standard MS medium contains 100 µM Fe. At the highest concentration, transcript levels were similar to those measured in plants grown on the standard. However, when Fe was limited, transcripts of OsYSL2, OsYSL9, and OsYSL15 were increased. The degree of induction was greater in shoots than in roots. In contrast, a reduction in Fe concentration did not significantly affect the expression of OsYSL16. Previous analysis with northern blots showed that OsYSL15 transcripts are not detected in the leaves of Fe-deficient rice plants (Koike et al., 2004; Inoue et al., 2009). This experimental inconsistency may have been due to differences in growing conditions, age of the plants, and genetic background. Here, we germinated rice seeds on agar medium containing different Fe concentrations and then grew the seedlings for 7 d prior to sampling. In contrast, Koike et al. (2004) and Inoue et al. (2009) reared their seedlings on an Fe-sufficient nutrient medium for 3 weeks, then transferred the plants to an Fe-deficient solution for 3 more weeks before sampling. Therefore, it is possible that their plants were not fully Fe limited at the sampling time. Furthermore, we used a different rice cultivar (Dongjin) whose genetic variation may have affected OsYSL15 expression under Fe deficiency. However, the expression of OsYSL2, which was induced by Fe deficiency in root and shoots, was similar to that described in a previous report (Koike et al., 2004).

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Real-time PCR analysis of OsYSL genes under different Fe concentrations. Seedlings were grown for 7 d on MS medium containing 0, 1, 10, 100, or 500 µM Fe. Each value is the average of three independent experiments. Transcript levels are represented by the ratio between mRNA levels of OsYSL2 (A), OsYSL9 (B), OsYSL15 (C), and OsYSL16 (D) and those of rice Actin1. The nomenclature of OsYSL genes in this study is the same as presented by Koike et al. (2004). Error bars indicate SD. Gene-specific primers are listed in Supplemental Table S2.

Expression Analysis of OsYSL15 Using the Promoter-GUS Fusion Molecule

To investigate the spatial and temporal expression patterns of OsYSL15, we generated transgenic plants carrying the 1.0-kb OsYSL15 promoter region that was fused to the uidA reporter gene. Histochemical GUS staining of 5-d-old seedlings showed that GUS activity was stronger in seedlings grown under Fe-deficient conditions (Fig. 2A , right) than in those in an Fe-sufficient scenario (Fig. 2A, left). Cross sections of the seminal roots cultured under the latter exhibited activity that was present mainly in the vascular cylinder (Fig. 2B). In response to Fe deficiency, GUS activity increased throughout the root tissues, including epidermis, exodermis, endodermis, cortex, and vascular cylinder (Fig. 2C). In leaves, activity was hardly detectable when the plants were grown on an Fe-supplemented medium (Fig. 2, D and F). When Fe was deficient, the OsYSL15 promoter became active in all tissues except the epidermal cells, suggesting a role for OsYSL15 in Fe distribution within the leaves (Fig. 2, E and G). These observations coincide with our previous results from quantitative real-time PCR analysis (Fig. 1C). However, results from our promoter-GUS analysis contrast with those previously reported by Inoue et al. (2009), where the OsYSL15 promoter was weakly active in companion cells and in some of the xylem parenchyma of Fe-sufficient leaves. Fe deficiency treatment did not change expression levels or localization. In our experiments, we used 5-d-old seedling plants grown in sufficient or deficient medium to determine OsYSL15 promoter activity. By comparison, Inoue et al. (2009) used 6-week-old plants to analyze GUS expression. This difference may explain these contradictory results.

View larger version (107K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Spatial expression patterns of the OsYSL15-GUS construct. A, Five-day-old seedlings grown on Fe-sufficient (Fe+) or Fe-deficient (Fe–) medium. B, Cross section of a 5-d-old seedling root grown under Fe-sufficient conditions. The analyzed region is at the differentiation zone (arrowhead in A). C, Cross section of a 5-d-old seedling root grown on Fe-deficient medium. The analyzed region is at the differentiation zone (arrowhead in A). D and E, Cross sections of midrib regions from leaves under Fe-sufficient (D) or Fe-deficient (E) conditions. The analyzed regions are indicated as arrowheads in A. F and G, GUS expression in leaf blades under Fe-sufficient (F) or Fe-deficient (G) conditions. H, Temporal and spatial expression patterns of OsYSL15-GUS fusion in spikelets at various developmental stages. Samples are as follows: 1, tetrad; 2, early young microspore; 3, late young microspore; 4, vacuolated pollen; 5, late pollen mitosis; 6, dehulled flower at late pollen mitosis stage. I, GUS activity in developing seeds at various stages. Samples are as follows: 1, 3 d after pollination (DAP); 2, 5 DAP; 3, 7 DAP; 4, 10 DAP; 5, 20 DAP. A, Anther; AR, aerenchyma; C, cortex; CE, chalazal end; E, embryo; ED, endodermis; EP, epidermis; EX, exodermis; G, glume; LO, lodicules; M mesophyll; P, palea; V, vascular bundle. Bars = 0.5 cm in A, 50 µm in B to G, 1 mm in H, and 0.5 mm in I.

OsYSL15 Is a Plasma Membrane-Localized Transporter

To determine the subcellular localization of OsYSL15, we prepared a DNA construct containing a fusion between OsYSL15 and GFP (Fig. 3 ). This OsYSL15-GFP construct was expressed transiently in onion (Allium cepa) epidermal cells. The PSORT program (http://psort.nibb.ac.jp) predicted that OsYSL15 would localize to the plasma membrane with high probability. We also used a control construct, which expressed a fusion protein between red fluorescent protein (RFP) and the Arabidopsis (Arabidopsis thaliana) proton ATPase2 (AHA2) that is localized to the plasma membrane (Fig. 3A; Kim et al., 2001). The DNA constructs, encoding OsSYL15-GFP and AHA2-RFP under the control of the cauliflower mosaic virus 35S promoter, were simultaneously delivered via particle bombardment (Fig. 3B). After 12 h of incubation, expression of the introduced genes was examined with a fluorescence microscope. The green fluorescent signal was detected at the plasma membrane coincident with the red signal driven by the AHA2-RFP (Fig. 3B). When GFP alone was expressed as a control, the protein was localized in the cytoplasm and nucleus (Fig. 3B). These results suggest that OsYSL15 is located at the plasma membrane.

View larger version (46K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Subcellular localization of OsYSL15:GFP in onion epidermal cells. A, Schematic diagrams of fusion constructs. P35S, Cauliflower mosaic virus 35S promoter; Tnos, nopaline synthase gene terminator. B, Onion cells cotransformed with OsYSL15:GFP and AHA2:RFP fusion constructs via particle bombardment. Fluorescent signals were examined 12 h after transfection. Data are representative of transformed cells. At least two independent transformation experiments were performed with each construct. "Merged" indicates that green and red signals were merged. Expression of GFP alone was analyzed as a control. Bar = 25 µm.

We tested whether OsYSL15 is capable of transporting Fe using the fet3fet4 yeast strain that is defective in Fe uptake (Dix et al., 1994). In this assay, the mutant strain was transformed with either the OsYSL15-expressing plasmid or the empty pYES6/CT vector, which served as a negative control. We utilized a β-estradiol-regulated expression system to control the level of OsYSL15 expression in yeast (Gao and Pinkham, 2000). Via this system, we achieved a tightly regulated off state. Viability of the strains was proved by growing them under permissive conditions (50 µM Fe citrate; Fig. 4, A and G ). To examine whether OsYSL15 can transport Fe³⁺, the strain containing the vector alone or else OsYSL15 was grown on a medium with FeCl₃ (Fig. 4B) or on one with FeCl₃ plus DMA (Fig. 4C). This experiment showed that OsYSL15 complemented yeast growth in the presence of FeCl₃ and DMA but not with FeCl₃ alone, thereby suggesting that OsYSL15 is able to transport Fe-PS complexes. When β-estradiol was withheld from the medium, the strain was unable to grow, demonstrating its dependence upon OsYSL15 expression (Fig. 4D). Functional complementation still occurred when a strong Fe²⁺ chelator, 4,7-biphenyl-1,10-phenanthroline-disulfonic acid (BPDS), was used to remove any residual Fe²⁺ from the Fe³⁺-DMA medium (Fig. 4E), thus supporting that OsYSL15 transports Fe³⁺.

View larger version (34K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Functional complementation of fet3fet4 yeast. DEY1453-derived yeast strains transformed with pGEV-TRP and constructs expressing OsYSL15 or empty pYES6/CT were grown on synthetic defined medium containing 50 µM Fe citrate (A and G); 10 µM FeCl3 with 40 nM β-estradiol (B); 10 µM FeCl3 with 10 µM DMA and 40 nM β-estradiol (C); 10 µM FeCl3 with 10 µM DMA without β-estradiol (D); 10 µM FeCl3 with 10 µM DMA, 10 µM BPDS, and 40 nM β-estradiol (E); 10 µM FeCl3, 10 µM DMA, and 10 µM BPDS without β-estradiol (F); 3 µM FeSO4 with 10 nM β-estradiol (H); 3 µM FeSO4 with 8 µM NA and 10 nM β-estradiol (I); or 3 µM FeSO4 with 8 µM NA without β-estradiol (J). Pairs of spots correspond to 10-fold and 100-fold dilutions of original cultures.

Disruption of OsYSL15 Results in Chlorotic Phenotypes under Fe Deficiency

To examine the role of OsYSL15 further, we isolated mutants in which the OsYSL15 gene was disrupted. From our rice flanking sequence tag database (An et al., 2003; Jeong et al., 2006), we identified two independent T-DNA knockout alleles. T-DNA was inserted into the second intron and second exon in osysl15-1 and osysl15-2, respectively (Fig. 5A ). T2 progeny were genotyped by PCR to obtain homozygous knockout plants and segregated wild-type siblings using gene-specific primers and a T-DNA primer (Fig. 5A). Reverse transcription (RT)-PCR analysis revealed the disruption of OsYSL15 expression in T-DNA homozygous plants, demonstrating that both are null alleles (Fig. 5B).

View larger version (63K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Isolation and characterization of OsYSL15 knockout mutant plants. A, Schematic diagram of OsYSL15 and insertion positions of T-DNA. Black boxes indicate seven exons, and connecting black lines represent introns. In line 2D10712, T-DNA was inserted into the second intron (osysl15-1) of OsYSL15; in line 3A10357, DNA was inserted into the second exon (osysl15-2) of OsYSL15. Horizontal arrowheads indicate primers (F1, R1, F2, and R2) used for genotyping T2 progeny. LB, Left border; RB, right border. B, RT-PCR analyses of OsYSL15 expression. OsYSL15-specific primers (Fw and Rv) were used with total RNA from leaves of osysl15-1, osysl15-2, or segregated wild-type (WT) seedlings grown under Fe deficiency. Rice Actin1 (OsAct1) was amplified as an internal control. C to E, Phenotype analyses of OsYSL15 knockout plants at seedling stage under Fe and Zn deficiencies. Homozygous and wild-type progeny of osysl5-1 and osysl15-2 were germinated and grown for 10 d on solid MS medium in the presence of Fe (100 µM) and Zn (30 µM) or in the absence of Fe (Fe–) or Zn (Zn–). Bottom panels show enlargements of second leaves. C, Wild-type and knockout mutant plants grown on MS control medium. D, Wild-type and knockout mutant plants grown on Fe-deficient medium. E, Wild-type and knockout mutant plants grown on Zn-deficient medium. Photographs were taken 10 d after germination. Bars = 5 cm. [See online article for color version of this figure.]

To evaluate whether the disruption of OsYSL15 affects Fe distribution, we measured Fe concentrations in shoots and roots at the seedling stage. When plants were grown in an Fe-sufficient medium, concentrations from osysl15-1 and osysl15-2 were reduced to 79% and 77% that of the wild type in the shoots and to 84% and 84% that of the wild type in the roots (Fig. 6A ). Under Fe deficiency, relative concentrations in osysl15-1 and osysl15-2 also were decreased to 79% and 75% in shoots and to 78% and 79% in roots, respectively (Fig. 6A). However, Zn concentrations in osysl15-1 and osysl15-2 were not significantly different from those in the wild-type plants (Fig. 6B). Levels of Cu and manganese (Mn) were also unchanged in shoots and roots (Supplemental Fig. S3, A and B).

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. A and B, Fe (A) and Zn (B) concentrations in shoots and roots from wild-type (WT) and osysl15 mutant plants grown on standard MS, Fe-deficient (Fe–), or Zn-deficient (Zn–) medium. At least two independent experiments were conducted for metal measurements, each using four plants. C and D, Fe concentrations in protoplasts and chloroplasts from wild-type and osysl15-1 plants grown on standard MS or Fe-deficient (Fe–) medium. E, Fe concentration in seeds from paddy-grown plants. F, Zn concentration in 10 pooled seeds, with four replicates prepared. Error bars indicate SE. Significant differences from the wild type were determined by Student's t test (* P < 0.05). Dw, Dry weight; Fw, fresh weight.

Promoter-GUS analysis showed that OsYSL15 was also active during seed development. Therefore, we postulated that disruption of OsYSL15 would affect Fe loading into the grains. In fact, those from osysl15-1 and osysl15-2 had 83% and 87% as much Fe, respectively, as seeds measured from the wild type (Fig. 6E), while Zn concentrations were similar for both homozygous knockout plants and the wild type (Fig. 6F). Levels of Cu and Mn were unchanged in mature seeds (Supplemental Fig. S3, C and D).

Effect of NO on Reversing the Chlorotic Phenotype of osysl15

NO is able to reverse the chlorotic phenotypes of two Fe-inefficient maize mutants, ys1 and ys3, both impaired in their Fe uptake (Graziano et al., 2002). Because our osysl15 mutants also showed a chlorotic phenotype in response to Fe deficiency, we evaluated whether NO could likewise rescue the deficiency symptoms of the mutants. Rice seeds were germinated and seedlings were grown for 10 d on Fe-deficient media containing various concentrations of sodium nitroprusside (SNP), an NO donor. NO-mediated increases in chlorophyll concentration were more prominent in osysl15-1 than in wild-type plants (Fig. 7A ; Supplemental Fig. S4C). Although 10 µM SNP was effective in elevating chlorophyll levels in the wild type, 50 µM SNP was needed to reverse chlorophyll deficiencies in the mutant (Fig. 7C).

View larger version (76K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Effect of NO on reversion of chlorosis in the osysl15-1 mutant. A, Seeds were germinated and plants grown on Fe-sufficient (Fe+) or Fe-deficient (Fe–) medium containing various concentrations of SNP. Photographs were taken from middle sections of second leaves at 10 d after treatment. For NO depletion, 100 µM CPTIO was added to growth medium. B, Phenotypes of 10-d-old plants grown on Fe-deficient medium containing 50 µM SNP. C, Inhibitory effect of CPTIO on NO-treated plants. WT, Wild type. Bars = 5 cm. [See online article for color version of this figure.]

NO treatment did not change the whole plant Fe concentration or enhance translocation of Fe from one organ to another (Supplemental Fig. S4D). Our results are consistent with the previous suggestion that NO improves the internal availability of Fe.

Expression Analysis of Fe Homeostasis-Related Genes in osyls15-1

NO plays a role in many different signaling pathways and affects the expression of numerous genes. To evaluate its influence on Fe homeostasis, we examined three NAS genes and two ferritin genes. Transcript levels of OsNAS1 and OsNAS2 were not detectable when Fe was sufficient but were markedly increased in response to Fe deficiency (Fig. 8A ). In wild-type plants, NO treatment abolished gene expression even at a low dose (i.e. 10 µM SNP). In the osysl15-1 mutant, however, expression of those two genes could be detected even when 25 µM SNP was supplied (Fig. 8A). Under NO treatment, CPTIO induced their expression in the mutants but not in the wild type (Fig. 8B). OsNAS3 was expressed in plants grown on an Fe-sufficient medium but was suppressed when Fe was limited. NO treatment upon Fe deficiency increased OsNAS3 expression in both wild-type and mutant plants (Fig. 8A), although to a relatively lesser extent in the latter.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Expression analysis for Fe homeostasis-related genes. RNA was prepared from 10-d-old shoots. A, Real-time PCR analysis of three rice NAS genes and two rice ferritin genes after SNP treatment. B, Expression analyses of OsNAS and Osferritin genes in plants grown on medium supplemented with 100 µM CPTIO, an NO inhibitor. Transcript levels are represented by the ratio between mRNA level for OsNAS or Osferritin genes and that for rice Actin1. Error bars indicate SD. WT, Wild type.

Overexpression of OsYSL15 Using the Rice Actin Promoter

We placed OsYSL15 cDNA in a sense orientation under the control of the rice Actin1 promoter, resulting in pGA2875 (Fig. 9A ). After generating transgenic plants, we studied constitutive expression of that gene using RNA samples prepared from leaves (Fig. 9B). Based on our quantitative real-time PCR analysis, we selected lines 2 (OX-2) and 6 (OX-6) for further examination. Fe and Zn concentrations were measured in their seeds (Fig. 9, C and D) via an atomic absorption spectrometer. Although Fe concentrations in seeds from both transgenic lines were increased compared with the wild type (Fig. 9C), Zn concentrations were not changed by overexpression of OsYSL15 (Fig. 9D). The levels of Mn and Cu in mature seeds of transgenic plants were similar to those of the wild type (Supplemental Fig. S3, C and D).

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Generation of transgenic plants overexpressing OsYSL15. A, Schematic diagram of the pGA2875 construct. OsYSL15 cDNA was placed between the rice Actin promoter (Pact) and the nopaline synthase terminator (Tnos). B, Quantitative real-time PCR analysis of overexpression transgenic plants using RNA from leaves. Error bars indicate SD. Transcript levels are represented by the ratio between mRNA level for OsYSL15 and that for rice Actin1. C and D, Fe and Zn concentrations in mature seeds from wild-type and OsYSL15-overexpressing (OX-2 and OX-6) plants grown in paddy fields. WT indicates segregated siblings of selected lines. Error bars indicate SE. * P < 0.05. Dw, Dry weight.

Expression of OsYSL15 was strongly induced by Fe deficiency and was decreased as the Fe concentration increased (Fig. 1). In testing the phenotypes of osysl15-1 knockout and OsYSL15-overexpressing plants, we observed that as the level of Fe rose, chlorophyll concentrations were increased in a dose-dependent manner (Fig. 10 ). Visual differences were documented by measuring chlorophyll concentrations (Fig. 10B). Under Fe-deficient conditions, the osysl15-1 knockout plants showed greater chlorosis, but that phenotype disappeared when higher Fe concentrations (at least 100 µM) were supplied. In OX-2 and OX-6 plants, this chlorotic phenotype was diminished at an Fe concentration of 10 µM or greater. These results indicate that OsYSL15 functions primarily when plants have a low availability of Fe.

View larger version (50K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Phenotype analyses of OsYSL15-overexpressing transgenic and osysl15-1 plants grown under different Fe concentrations. A, Representative photographs of second leaves. WT and T/T indicate segregated wild-type and homozygous plants from selected lines. B, Chlorophyll concentrations from plants (n = 4) grown at different Fe concentrations. Error bars indicate SE. * P < 0.05. [See online article for color version of this figure.]

To examine how the disruption or overexpression of OsYSL15 might influence plant architecture and grain yields, we cultivated transgenic seedlings along with their segregated wild-type siblings in the field. All knockout plants as well as OX-2 and OX-6 plants flowered about 10 d later than their wild-type segregants. Whereas transgenic plant heights were significantly reduced (Fig. 11 ), tiller numbers did not change (Supplemental Table S1). Moreover, although fewer total spikelets were counted on the knockout mutants and overexpression plants, their grain yields were not significantly different from those of the wild type (Supplemental Table S1). We also measured Fe, Zn, Mn, and Cu concentrations in wild-type and mutant flag leaves, sampling for uniformity after flowering. Disruption of OsYSL15 resulted in reduced Fe levels in flag leaves, while its overexpression increased those values in flag leaves (Fig. 11E). However, Zn, Mn, and Cu concentrations in the flag leaves were unaffected by either disruption or overexpression of OsYSL15 (Fig. 11F; Supplemental Fig. S3, F and G).

View larger version (78K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 11. Phenotypes of OsYSL15 knockout and overexpressing transgenic plants grown in the paddy field. Representative photographs were taken after plants flowered. A and B, Comparisons among wild-type (WT), osysl15-1, and osysl15-2 plants. C and D, Comparison of wild-type and OsYSL15-overexpressing (OX-2 and OX-6) plants. For the photographs, transgenic plants were transferred to pots from the paddy fields. Bars = 10 cm. E and F, Fe (E) and Zn (F) concentrations in flag leaves, with each measured from four flag leaves per line sampled after flowering. Error bars indicate SE. * P < 0.05. [See online article for color version of this figure.]

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Disruption of OsYSL15 resulted in a 20% reduction in Fe concentration under both sufficient and deficient conditions. Although no altered phenotypes were visible when Fe supplies were adequate, severe chlorosis occurred in the osysl15 mutants when Fe was limited. This suggests that OsYSL15 is important for distribution into the chloroplast, as was correlated with a great reduction in chloroplast Fe concentrations. Therefore, how OsYSL15 affects Fe distribution into chloroplasts needs to be investigated.

The disruption or overexpression of OsYSL15 was manifested by shorter plants and alterations in their architecture. Therefore, because only the concentration of Fe varied, Fe homeostasis must play an important role in growth and development. Because the Fe concentration was altered by such disruption or overexpression, the physiological balance of metal ions was disturbed, resulting in defective growth in the field. When IRT1 is ectopically expressed, transgenic plants show no visible morphological changes (Connolly et al., 2002). Although IRT1 mRNA is expressed constitutively, IRT1 protein is present only in Fe-limited roots, indicating posttranscriptional regulation of IRT1 (Vert et al., 2002). Because our OsYSL15 overexpressors accumulated more Fe and had altered growth, we can assume that OsYSL15 protein levels are higher in the overexpression lines than in the wild types. However, we do not know that posttranscriptional regulation is occurring as AtIRT1. Unfortunately, we were unable to measure the level of OsYSL15 protein due to a lack of available antibodies.

Seedlings of the maize ys1 mutant, which is defective in the uptake of Fe³⁺-PS complexes, experience severe Fe deficiency chlorosis (yellowing between the veins) and, ultimately, mortality, indicating that such uptake is an essential process for that species (Walker and Connolly, 2008). Here, however, mutation in OsYSL15 did not produce lethal phenotypes, although those plants were shorter and contained less Fe, probably because of gene redundancy. Three additional rice genes are highly homologous to maize YS1. Among them, induction patterns for OsYSL2 and OsYSL9 by Fe deficiency are quite similar to those for OsYSL15. Rice also has efficient Fe²⁺ uptake systems, in contrast to other grasses (Ishimaru et al., 2006).

NO is a bioactive molecule, playing important roles in many physiological processes, including determining Fe availability within a plant (Graziano et al., 2002). Here, we used osysl15 mutants to evaluate the effect of NO in rice. By treating with SNP, an NO donor, we were able to partially reverse their chlorotic phenotype. An NO scavenger, CPTIO, abolished that protective effect. However, NO treatment did not increase total Fe content within a plant. These results support that NO increases Fe availability in rice, as observed from other organisms. NO stimulates the accumulation of both ferritin mRNA and protein in Arabidopsis (Murgia et al., 2002). We observed similar positive effects on rice ferritin mRNA levels. In contrast, expression of two NAS genes that was induced by Fe deficiency was inhibited by NO. Therefore, we conclude that there are alternative ways to alleviate the stresses associated with diminished supplies of Fe.

Quantitative real-time PCR and promoter-GUS analyses have indicated that OsYSL15 is strongly induced by lower Fe levels. Two cis-acting elements, IDE1 and IDE2, synergistically mediate Fe deficiency-induced gene expression in tobacco (Nicotiana tabacum; Kobayashi et al., 2003). Sequences similar to IDE1 and IDE2 are found in the promoter regions of OsIRT1, OsNAS1, and OsNAS2, which are induced by Fe deficiency (Kobayashi et al., 2003, 2005). The rice basic helix-loop-helix protein OsIRO2, an essential regulator of the genes involved in Fe uptake under deficient conditions, also contains putative IDE sequences in its promoter region (Ogo et al., 2006). Therefore, the regulatory networks mediated by IDE elements during Fe deficiency are apparently conserved between dicot and monocot species. The promoter region of OsYSL15 has two putative IDE sequences, at –249 and –593 bp from the translation initiation site. Our OsYSL15 promoter-GUS construct contained these two putative IDE-like elements, and GUS expression was induced by Fe deficiency. Further research is necessary to verify whether these elements are indeed responsible for that deficiency response. OsYSL15 is expressed strongly in OsIRO2-overexpressing transgenic plants (Ogo et al., 2007), suggesting that the former functions downstream of the latter.

OsYSL15 overexpression was positive in raising the Fe concentration in our seeds and vegetative tissues, albeit with some side effects. This presents the possibility that OsYSL15 can be used for enhancing Fe levels in rice grains, perhaps via targeted expression with seed-specific promoters.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Growth

Wild-type transgenic rice (Oryza sativa ‘Dongjin’) and seeds were surface sterilized and germinated on an MS solid medium supplemented with 0, 1, 10, 100, or 500 µM Fe³⁺-EDTA. Shoot and root samples from 7-d-old seedlings were frozen with liquid nitrogen. SNP (10–100 µM) was used as an NO donor, and 100 µM CPTIO served as an NO scavenger. Transgenic plants were transplanted and grown to maturity in paddy fields located at Pohang University of Science and Technology (36° N). The field tests were performed twice, in 2007 and 2008.

RNA Isolation and RT-PCR Analysis

Total RNA was obtained from each tissue type with an RNA isolation kit (Tri Reagent; MRC). For cDNA synthesis, we used 2 µg of total RNA as template and M-MLV reverse transcriptase (Promega) in a 25-µL reaction mixture. RT-PCR was performed in a 50-µL solution containing a 1-µL aliquot of the cDNA reaction, 0.2 µM gene-specific primers, 10 mM deoxyribonucleotide triphosphates, and 1 unit of rTaq DNA polymerase (Takara Shuzo). PCR products were separated by electrophoresis on a 1.2% agarose gel. Quantitative real-time PCR was performed with a Roche LightCycler II as described previously (Han et al., 2006). The Actin1 mRNA levels were used to normalize the expression ratio for each gene. Changes in expression were calculated via the _Ct method. The gene-specific primers are listed in Supplemental Table S2.

Yeast Functional Complementation

RNA was isolated from the roots of 30-d-old rice cv Nipponbare. cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen) with oligo(dT) primers. RT-PCR was performed with PfuTurbo DNA polymerase (Stratagene) and primers YF (5'-TGCAGCGAATTCTCGAGCAGCTAAGCGAGATCGAC-3') and YR (5'-TTCTAGAGCGGCCGCCTGCAATCCTCCACCCATGAAAT-3'), which contained EcoRI and NotI sites (underlined sequences) for cloning, respectively. The resulting product was ligated into the NotI/EcoRI-digested pYES6/CT vector. Saccharomyces cerevisiae strain DEY1453 (MATa/MATa ade2/ADE2 can1/can1 his3/his3 leu2/leu2 trp1/trp1 ura3/ura3 fet3-2::HIS3/fet3-2::HIS3 fet4-1::LEU2/fet4-1::LEU2) was transformed with pGEV-Trp (Gao and Pinkham, 2000) together with pYES6/CT or pYES6/CT expressing OsYSL15.

For complementation assays, synthetic dextrose (SD)-Trp medium was made with an Fe-free yeast nitrogen base buffered with 25 mM MES at pH 5.7 for plates containing Fe²⁺ or at pH 6.0 for plates containing Fe³⁺. To prepare our Fe²⁺ assay, the following were added, in order, to the center of each empty plate: 125 µL of 200 mM ascorbic acid, 7.5 µL of freshly prepared 10 mM Fe₂SO₄, and 20 µL of 10 mM NA. This solution was mixed briefly and then incubated at room temperature for 10 min to allow complex formation. Afterward, 25 mL of molten SD-Trp with 10 µg mL^–1 blasticidin was added, which was then allowed to solidify. To prepare for our Fe³⁺ assay, 34 µL of 7.4 mM FeCl₃ and 25 µL of 10 mM DMA were placed in the center of each empty plate and incubated at room temperature for 10 min. We then added 25 mL of molten SD-Trp with 10 µg mL^–1 blasticidin to the plates before solidification. To prepare plates with 10 mM BPDS, 25 µL was incorporated just prior to the addition of the molten medium.

Suspensions were prepared from 3-d-old yeast colonies, which were removed from the plates and suspended in sterile water before the optical density at 550 nm of the resulting suspension was measured. After that value was brought to 0.1, serial dilutions (1:10, 1:100, 1:1,000, and 1:10,000) of the suspension were prepared, and 7 µL of each dilution was spotted on the plates. They were then grown at 28°C for 3 d.

Generation of the OsYSL15 Promoter-GUS Fusion Molecule and GUS Assay

Genomic sequences (–1,000 to –1 bp from the translation initiation site) containing the promoter region of OsYSL15 were amplified by PCR using two primers (pf, 5'-AAAAGCTTAGCATGTCTCCAGATTCTCCAT-3'; and pr, 5'-AAGGATCCGCGGCGGCGGCGGCGTCGATCTC-3'). This fragment was connected to a GUS-NOSt cassette (derived from pBI101.2) and ligated into pCAMBIA1302, resulting in pGA2866. This plasmid was transferred to Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method (An et al., 1988), and transgenic plants carrying the above construct were generated via Agrobacterium-mediated cocultivation (Lee et al., 1999). Histochemical GUS staining of those transgenic plants was performed according to the method reported by Dai et al. (1996). Ten-micrometer sections were prepared as described previously by Jung et al. (2005) and were observed with a microscope (Nikon) under bright-field illumination.

Subcellular Localization of the OsYSL15-GFP Fusion Protein in Onion Epidermal Cells

Full-length OsYSL15 cDNA was PCR amplified with the primer pair gf (5'-AATCTAGAGTTTCTTTCTTGTCCTCGTGGT-3') and gr (5'-AAGGATCCAGCTTCCAGGCGTAAACTTCATGC-3'). These primers contained XbaI or BamHI sites (underlined sequences) to facilitate cloning of the amplified cDNA. After sequence analysis, the OsYSL15 cDNA was cloned into the XbaI and BamHI sites of the 326-GFP vector (Lee et al., 2001). The AHA2-RFP fusion molecule under the control of the cauliflower mosaic virus 35S promoter was obtained from Inhwan Hwang (Pohang University of Science and Technology). Constructs were introduced into onion (Allium cepa) epidermal cells by particle bombardment using the Biolistic PDS-1000/He particle delivery system (Bio-Rad). At 12 h after transformation, expression of GFP and RFP was monitored with a fluorescence microscope and two filters (Axioplan2; Carl Zeiss).

Isolation of OsYSL5 Loss-of-Function Plants

Two putative OsYSL15 knockout mutants were isolated from our rice flanking sequence tag database (http://www.postech.ac.kr/life/pfg). T2 progeny of the primary mutants were grown to maturity to amplify their seeds. Genotyping of these progeny was determined by PCR using three primers. These included the following: for osysl15-1 (line 2D-10712), two specific primers (F1, 5'-GCCTTTCTTCCCTTAATTTGATCCAC-3'; and R1, 5'-CTTAACAAATCTATACTGCTTT-3') and one T-DNA-specific primer (LB; 5'-ACGTCCGCAATGTGTTATTAA-3'); for osysl15-2 (line 3A-10357), two specific primers (F2, 5'-ATAGGCAGAGGGTTCCATTT-3'; and R2, 5'-AGCCACCTCACACAAGAGAG-3') and a T-DNA-specific primer (LB; 5-ACGTCCGCAATGTGTTATTAA-3'). Afterward, transcript levels for OsYSL15 were determined by RT-PCR using cDNA prepared from the leaves of 10-d-old seedlings grown under Fe deficiency.

Generation of the OsYSL15-Overexpressing Construct

To create our OsYSL15-overexpressing construct, the full-length cDNA sequence of OsYSL15 was amplified by a primer pair (FL, 5'-AATCTAGAGTTTCTTTCTTGTCCTCGTGGT-3'; and RL, 5'-AACTCGAGACCTCTTAGCTTCCAGGCGTAA-3'). The PCR product was cloned into XbaI and XhoI sites between the rice Actin1 promoter (McElroy et al., 1990) and the T7 terminator of the binary vector pGA1671, thereby generating pGA2875 (Jeon et al., 2000). Transformation of this plasmid into Agrobacterium and the generation of transgenic plants were as described previously (Lee et al., 1999).

Preparation of Protoplast and Chloroplast

Mesophyll protoplasts were prepared as described previously by Moon et al. (2008). Briefly, the third leaves were harvested and dissected from 10-d-old seedlings grown on either MS or Fe-deficient medium. The materials were digested in an enzyme solution (1.5% cellulase RS, 0.3% macerozyme, 0.1% pectolyase, 0.6 M mannitol, 10 mM MES, 1 mM CaCl₂, and 0.1% [w/v] bovine serum albumin) for 4 h at 26°C with gentle agitation (50–75 rpm). KMC solution (117 mM KCl, 82 mM MgCl₂, and 85 mM CaCl₂) was added afterward. Protoplasts were sorted from the debris through a nylon mesh (20 µm), collected by centrifuging at 1,000 rpm for 2 min, and resuspended in EP3 solution (70 mM KCl, 5 mM MgCl₂, 0.4 M mannitol, and 0.1% MES, pH 5.6). Chloroplasts were isolated according to the published protocol (Triboush et al., 1998). Five grams of leaves from 10-d-old seedlings was homogenized in a mortar with 20 mL of STE buffer (400 mM Suc, 50 mM Tris, pH 7.8, 20 mM EDTA-Na₂, 0.2% bovine serum albumin, and 0.2% β-mercaptoethanol). After the homogenate was filtered through a 50-µm nylon mesh, the extract was centrifuged at 1,000 rpm. The supernatant was then centrifuged at 4,000 rpm, and this resuspended pellet was centrifuged again at 4,000 rpm to prepare the chloroplast fraction.

Measurement of Plant Growth and Metal Concentrations

Seeds of the wild type and mutant were germinated, and plants were then grown for 10 d on a solid medium containing MS salts supplemented with different concentrations of Fe³⁺-EDTA. Their chlorophyll concentrations were measured as described previously (Lee et al., 2005). Briefly, 0.1 g of leaf samples was harvested and the chlorophyll was extracted with 1 mL of 80% acetone. After homogenization, the samples were incubated for 15 min and spun at 15,000g for 10 min before an aliquot of the supernatant fraction was taken to measure A₆₆₃ and A₆₄₃ with a spectrophotometer. Chlorophyll concentrations, including chlorophyll a and b, were determined according to the method of Arnon (1949), and metal concentrations were measured as described previously by Kim et al. (2002). Seeds and shoot and root portions were dried for 2 d at 70°C before those samples were weighed. Afterward, they were digested in 1 mL of 11 N HNO₃ for 3 d at 200°C. Following dilution, their metal concentrations were determined by an atomic absorption spectrometer (SpectrAA-800; Varian).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers number AB190923.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Expression analyses of OsYSL genes that were constitutive under different iron concentrations.

Supplemental Figure S2. Quantification of wild type and osysl15 mutants grown on control MS (100 µM Fe and 30 µM Zn), Fe-deficient (Fe–), or Zn-deficient (Zn–) medium for 10 d following germination.

Supplemental Figure S3. Mn and Cu concentrations in wild-type and transgenic plants.

Supplemental Figure S4. Characterization of wild-type and osysl15-1 mutant plants grown with various concentrations of SNP.

Supplemental Table S1. Characterization of osysl15 mutants grown in the paddy field.

Supplemental Table S2. Primers used for RT-PCR analysis.

	ACKNOWLEDGMENTS

 
We thank Inhwan Hwang for providing AtAHA2:RFP, In-Soon Park and Kyungsook An for plant transformation, Jongdae Kyung for technical assistance with the atomic absorption spectrometer measurements, Changduk Jung for growing plants, and Priscilla Licht for critical reading of the manuscript.

Received January 8, 2009; accepted April 14, 2009; published April 17, 2009.

	FOOTNOTES

 
¹ This work was supported by the Crop Functional Genomic Center, 21st Century Frontier Program (grant no. CG1111), the Biogreen 21 Program of the Rural Development Administration (grant no. 20070401–034–001–007–03–00), the National Research Laboratory Program of the Ministry of Science and Technology (grant no. M10600000270–06J0000–27010), the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (grant no. 2005–01072 to E.L.W.), and the National Science Foundation (grant no. DB10701119 to M.L.G.).

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: Gynheung An (genean{at}postech.ac.kr).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.135418

^* Corresponding author; e-mail genean{at}postech.ac.kr.

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