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J Biol Chem
2011 Feb 18;2867:5446-54. doi: 10.1074/jbc.M110.180026.
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Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants.
Nozoye T
,
Nagasaka S
,
Kobayashi T
,
Takahashi M
,
Sato Y
,
Sato Y
,
Uozumi N
,
Nakanishi H
,
Nishizawa NK
.
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Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released (14)C-labeled deoxymugineic acid but not (14)C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.
FIGURE 1. Efflux activity of TOM1-, HvTOM1-, ENA1-, ENA2-, and AK068840-encoded proteins in X. laevis oocytes. Time-dependent efflux of [14C]DMA and [14C]NA is shown. Oocytes expressing TOM1, HvTOM1, ENA1, ENA2, or AK068840 and control oocytes were injected with water or with [14C]DMA (A and B) or [14C]NA (C and D), and the efflux of 14C was measured for 1–24 h. Values are expressed as the percentage of the total radioactivity injected. Data shown represent means ± S.D. (error bars; n = 3).
FIGURE 2. Subcellular localization of TOM1 in onion epidermal cells (A–C) and in rice roots (D–F). A and D, differential interference contrast image. B and E, fluorescence image. C and F, overlay.
FIGURE 3. Characters of TOM1 family. A, amino acid sequence of HvTOM, TOM1, TOM2, and TOM3. The 12 putative membrane-spanning domains predicted using the TM-pred program are shown in upperlines. B, location of TOM family on the chromosome 11.
FIGURE 4. Phylogenetic tree of plant TOM transporters. Os, rice; Hv, barley; Zm, maize; At, Arabidopsis; Sl, tomato; Ta, wheat. Scale bar, 0.1 substitution/site.
FIGURE 5. Expression of TOM1. A, regulation of TOM1 mRNA levels by Fe availability in rice. The upper panel shows the Northern blot analysis, and the lower panel shows the quantitative RT-PCR analysis of TOM1. +, Fe-sufficient; −, Fe-deficient. In the Northern blot analysis, ethidium bromide-stained rRNA is shown as a loading control. In the quantitative real time PCR analysis, the values were normalized with the expression of OsActin1 and represent the mean ± S.D. (error bars) from three reactions. B, diurnal changes in TOM1 expression in Fe-deficient rice roots. L, light; D, dark. C, diurnal changes in HvTOM1 expression in Fe-deficient (−) and Fe-sufficient (+) barley roots. L, light; D, dark.
FIGURE 6. Tissue distribution of TOM1 protein expression in rice during vegetative stage (A–D), seed development (E), and germination (F) as shown by GUS staining. A, Fe-sufficient root cross-section. B, Fe-deficient root cross-section. C, Fe-deficient leaf sheath cross-section. D, Fe-deficient leaf cross-section. E, seeds before anthesis; after fertilization; 5, 8, 20, and 30 days after flowering (DAF), and immediately before full maturation. F, fully mature seeds (0d) and germinating rice seeds 1–3 days after sowing.
FIGURE 7. In vivo function of TOM1 in rice. A, Northern blot analysis in Fe-deficient roots of NT plants; plants overexpressing TOM1 (OXOs) or HvTOM1 (OXHv); and plants with RNAi-repressed TOM1 expression (RNAi). B, SPAD values (chlorophyll content) of the newest and oldest leaves of OXOs, OXHv, RNAi, and NT plants during progression of Fe deficiency stress (0 days) and 12 days later. Error bars represent S.E. (n = 3–6). C, Fe, Zn, and Cu contents in OXOs, OXHv, RNAi, and vector control (VC) seeds. Error bars represent S.D. *, significant difference (p < 0.01) by Student's t test.
FIGURE 8. Analysis of levels of endogenous DMA in and DMA secretion from Fe-deficient OXOs, OXHv, RNAi, and NT plant roots after a 6-h exposure to light. A, secreted DMA. B, endogenous DMA. C, fraction of the total DMA synthesized in the root that was secreted. Values shown represent the means of three replicates. Error bars represent S.D. *, significant difference (p < 0.01) by Student's t test.
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