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, 24 (5), 2155-67

Nramp5 Is a Major Transporter Responsible for Manganese and Cadmium Uptake in Rice

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Nramp5 Is a Major Transporter Responsible for Manganese and Cadmium Uptake in Rice

Akimasa Sasaki et al. Plant Cell.

Abstract

Paddy rice (Oryza sativa) is able to accumulate high concentrations of Mn without showing toxicity; however, the molecular mechanisms underlying Mn uptake are unknown. Here, we report that a member of the Nramp (for the Natural Resistance-Associated Macrophage Protein) family, Nramp5, is involved in Mn uptake and subsequently the accumulation of high concentrations of Mn in rice. Nramp5 was constitutively expressed in the roots and encodes a plasma membrane-localized protein. Nramp5 was polarly localized at the distal side of both exodermis and endodermis cells. Knockout of Nramp5 resulted in a significant reduction in growth and grain yield, especially when grown at low Mn concentrations. This growth reduction could be partially rescued by supplying high concentrations of Mn but not by the addition of Fe. Mineral analysis showed that the concentration of Mn and Cd in both the roots and shoots was lower in the knockout line than in wild-type rice. A short-term uptake experiment revealed that the knockout line lost the ability to take up Mn and Cd. Taken together, Nramp5 is a major transporter of Mn and Cd and is responsible for the transport of Mn and Cd from the external solution to root cells.

Figures

Figure 1.
Figure 1.
Expression Pattern of Nramp5. (A) Relative expression in various tissues at different growth stages. Rice was grown in a paddy field until ripening and various tissues were sampled. (B) Root spatial expression of Nramp5. RNA was extracted from the root tip (0 to 1 cm) or basal root region (1 to 2 cm). Asterisks indicate significant difference from the wild type at **P < 0.01 by Student’s t test. (C) Response of Nramp5 expression to metal deficiency. Rice was cultivated in a nutrient solution with (control) or without Zn, Fe, Mn, or Cu for 1 week. The expression level was determined by quantitative real-time RT-PCR. Expression relative to the root at flowering stage (A), root tip (B), and root in control condition (C) are shown. HistoneH3 and Actin were used as internal standards. Data are means ± sd of three biological replicates. Asterisks indicate significant difference from control condition at *P < 0.05 by Dunnett’s test. [See online article for color version of this figure.]
Figure 2.
Figure 2.
Cellular and Subcellular Localization of Nramp5. (A) to (D) Immunostaining of roots of wild-type ([A], [C], and [D]) and knockout line nramp5 (B) rice. (C) and (D) High-magnification image of the endodermis (en) and exodermis (ex), respectively, costained with DAPI. Immunostaining was performed using Os Nramp5 antibody. Red represents signal from the antibody and cyan from cell wall autofluorescence. Nuclei were stained with DAPI (arrow). Bars = 20 μm. (E) and (F) Subcellular localization of Nramp5-GFP (E) and GFP alone (F) in the epidermal cells of onion. Bars = 100 μm.
Figure 3.
Figure 3.
Phenotypic Analysis of the Nramp5 Knockout Line. (A) and (B) Growth of wild-type (WT) rice and the knockout line nramp5. (A) Shoot dry weight. (B) Root dry weight. (C) and (D) Mineral concentration of the shoots (C) and roots (D). Both wild-type rice and the knockout line were grown in a nutrient solution containing 0.5 μM MnCl2, 10 μM FeSO4, and 0.1 μM CdSO4 for 3 weeks. The shoots and roots were harvested and subjected to mineral analysis by inductively coupled plasma–mass spectrometer. Data are means ± sd of three biological replicates. Asterisks indicate significant difference from the wild type at *P < 0.05 and **P < 0.01 by Student’s t test.
Figure 4.
Figure 4.
Growth, Yield, and Mineral Analysis of the Nramp5 Knockout Line Grown in Soil. (A) Straw dry weight. WT, the wild type. (B) Grain yield. (C) and (D) Metal concentration of the straw (C) and brown rice (D). Both wild-type rice and the knockout line were grown in soil for 4 months. Data are means ± sd of three biological replicates. Asterisks indicate significant difference from the wild type at *P < 0.05 and **P < 0.01 by Student’s t test.
Figure 5.
Figure 5.
Phenotypic Analysis of the Nramp5 Knockdown Line. (A) Expression level of Nramp5 in the roots of wild-type (WT) rice (cv Nipponbare) and three independent RNAi lines. PCR was run for 26 cycles. (B) Shoot dry weight. (C) and (D) Concentration of Mn (C) and Fe (D) in the shoots. Both wild-type rice and RNAi lines were cultivated in a nutrient solution containing 0.5 µM MnCl2 and 10 μM FeSO4 for 4 weeks. DW, dry weight. (E) Mineral analysis of the Nramp5 knockdown line grown in soil. Data are means ± sd of three biological replicates. Asterisks indicate significant difference from the wild type at *P < 0.05 by Dunnett’s test.
Figure 6.
Figure 6.
Rescue of the Growth Phenotype of the Nramp5 Knockout Line by Mn Supplementation. (A) to (F) Growth of wild-type rice (left) and the knockout line (right) grown in a nutrient solution containing 0.1 ([A] and [D]), 0.5 ([B] and [E]), or 5 ([C] and [F]) µM MnCl2 and 2 μM FeSO4 for 3 weeks. (A) to (C) Whole seedlings, (D) to (F) The youngest leaf. (G) and (H) Dry weight of shoots (G) and roots (H). Data are means ± sd of three biological replicates. Different letters indicate significant difference at P < 0.05 by Tukey’s test. WT, the wild type.
Figure 7.
Figure 7.
Concentration of Mn and Fe in the Shoots and Roots at Different Mn Supplies. (A) and (B) Concentration of Mn (A) and Fe (B) in the shoots of wild-type (WT) and nramp5 plants. (C) and (D) Concentration of Mn (C) and Fe (D) in the roots of wild-type and nramp5 plants. Both wild-type rice and the knockout line were cultivated in a nutrient solution containing 0.1, 0.5, or 5 µM MnCl2 and 2 μM FeSO4 for 3 weeks. Data are means ± sd of three biological replicates. Asterisks indicate significant difference from the wild type at **P < 0.01 by Student’s t test.
Figure 8.
Figure 8.
Growth and Metal Concentration at Different Concentrations of Fe. (A) and (B) Dry weight of shoots (A) and roots (B). Different letters indicate significant difference at P < 0.05 by Tukey’s test. WT, the wild type. (C) and (D) Concentration of Fe (C) and Mn (D) in the shoots. (E) and (F) Concentration of Mn (E) and Fe (F) in the roots. Both the wild-type rice and knockout line were grown in a nutrient solution containing 0.1, 2, or 10 µM FeSO4 for 3 weeks. Data are means ± sd of three biological replicates. In (C) to (F), asterisks indicate significant difference from the wild type at *P < 0.05 and **P < 0.01 by Student’s t test.
Figure 9.
Figure 9.
Short-Term Uptake of Mn and Cd by Rice Roots. (A) and (C) Uptake of Mn (A) and Cd (C) at 25 and 4°C. DW, dry weight; WT, the wild type. (B) and (D) Net uptake of Mn (B) and Cd (D). The uptake was determined by exposing seedlings of both the wild-type rice and knockout line to different Mn or Cd concentrations at 25 and 4°C for 30 min. Net uptake was calculated by subtracting the apparent uptake at 4°C from that at 25°C. Data are means ± sd of three biological replicates.

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