Characterizing the role of rice NRAMP5 in Manganese, Iron and Cadmium Transport

Abstract

Metals like manganese (Mn) and iron (Fe) are essential for metabolism, while cadmium (Cd) is toxic for virtually all living organisms. Understanding the transport of these metals is important for breeding better crops. We have identified that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice. OsNRAMP5 expression was restricted to roots epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem. OsNRAMP5 localized to the plasma membrane, and complemented the growth of yeast strains defective in Mn, Fe, and Cd transport. OsNRAMP5 RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. The suppression of OsNRAMP5 promoted Cd translocation to shoots, highlighting the importance of this gene for Cd phytoremediation. These data reveal that OsNRAMP5 contributes to Mn, Cd, and Fe transport in rice and is important for plant growth and development.

Figure 1. Metal concentrations of rice plants grown in the presence of Cd.

Shoot and root metal concentrations under control conditions (Ctrl.) or in the presence of 10 µM Cd (+Cd). (a, e) Mn, (b, f) Fe, (c, g) zinc, and (d, h) copper concentrations. Error bars represent the SD. Columns bars followed by different letters are statistically different according to analysis of variance followed by Student-Newman-Keuls (SNK) test (a, p = 0.0034; b, p = 0.0074; c, p = 0.0000; d, p = 0.0000; e, p = 0.0017; f, p = 0.0069; g, p = 0.0005; h, p = 0.0010); n = 3.

Figure 2. OsNRAMP5 is expressed in rice roots.

(a–c) Expression pattern of OsNRAMP5 in rice grown hydroponically under Mn-deficient (-Mn) (a), Fe-deficient conditions (-Fe) (b), and in the presence of 10 µM Cd (+Cd) (c) compared to normal nutrient conditions (Ctrl.). (d–f) OsNRAMP5 promoter GUS analysis in rice roots. Longitudinal section (d). Transverse section (e). Enlargement of part of the stele (f). MXII, metaxylem II; MXI, metaxylem I; PX, protoxylem. Scale bar = 400 μm for (d), and 50 μm for (f). Error bars represent the SD. Columns bars followed by different letters are statistically different according to analysis of variance followed by SNK test (p = 0.0034); n = 3.

Figure 3. OsNRAMP5 contributes to Mn, Fe and Cd transport.

(a–d) Green fluorescent protein (GFP) fluorescence in onion epidermal cells observed by confocal laser scanning microscopy (a), OsNRAMP5-sGFP fluorescence (b), fluorescence of FM4-64 and (c) overlay of FM4-64 and OsNRAMP5-sGFP. (d) Fluorescence of sGFP only. Scale bar = 50 μm. (e–g) Serial dilutions of yeast cells for Δsmf (Mn uptake mutant) (e), Δfet3fet4 (Fe uptake mutant) (f), Δycf1 (Cd-sensitive mutant) (g), transformed with empty vector (V.C.), OsNRAMP1 or OsNRAMP5.

Figure 4. Characterization of OsNRAMP5i and OsNRAMP5 OX lines.

(a, b) OsNRAMP5 transcription in roots (a) and shoots (b) of wild type, OsNRAMP5i (T5i-1, T5i-2), and OsNRAMP5OX (OX1, OX2) plants (cultivar Tsukinohikari) grown in control hydroponic culture. (c, d) Phenotypes of wild type and T5i plants grown under control (c) and Mn-deficient conditions (d). Scale bars = 10 cm. Root length (e), shoot length (f), SPAD value (g), root (h) and shoot (i) Mn concentrations, root (j) and shoot (k) Fe concentrations of plant grown under control conditions or Mn-deficient (-Mn) conditions. Error bars represent the SD. Columns bars followed by different letters are statistically different according to analysis of variance followed by SNK test (a, p = 0.0013; b, p = 0.0003; e, p = 0.0017; f, p = 0.0009 (Ctrl.) & p = 0.0006 (-Mn); g, p = 0.0001; h, p = 0.0000 (Ctrl.) & p = 0.0067 (-Mn); i, p = 0.0000 (Ctrl.) & p = 0.0004 (-Mn); j, p = 0.0095; k, p = 0.0000; n = 3.

Figure 5. Xylem sap metal concentrations in OsNRAMP5i plants.

Xylem Mn (a), Fe (b), and Ca (c) concentrations in T5i plants grown under control or Mn-deficient (-Mn) conditions. Error bars represent the SD. Columns bars followed by different letters are statistically different according to analysis of variance followed by SNK test (a, p = 0.0001 (Ctrl.) & p = 0.0010 (-Mn); b, p = 0.0000 (Ctrl.) & p = 0.0001 (-Mn); n = 3.

Figure 6. Suppression of OsNRAMP5 leads to high Cd in rice shoots.

Shoot (a) and root (e) Mn; shoot (b) and root (f) Fe; shoot (c) and root (g) Cd concentrations and shoot (d) and root (h) Cd content in T5i and OX plants grown under 10 µM Cd conditions. Error bars represent the SD. Columns bars followed by different letters are statistically different according to analysis of variance followed by SNK test (a, p = 0.0004; c, p = 0.0005; d, p = 0.0001; e, p = 0.0067; g, p = 0.0017; h, p = 0.0001); n = 3.

Figure 7. OsNRAMP5i Anjana Dhan for Cd phytoremediation.

(a) Phenotypes of four-week-old wild type and OsNRAMP5i (A5i-2) Anjana Dhan plants transferred to a nutrient solution containing 10 µM CdCl₂ and cultivated for two weeks. Scale bars = 10 cm. (b) OsNRAMP5 transcripts in the roots of wild type and OsNRAMP5i (A5i-1, A5i-2, A5i-3, A5i-4) Anjana Dhan plants grown in normal nutrient hydroponic culture. Shoot Mn (c), Fe (d) concentration, shoot dry weight (e), shoot Cd concentration (f) and Shoot Cd content (g) of wild type and A5i plants grown under 10 μM Cd condition. Error bars represent the SD. Columns bars followed by different letters are statistically different according to analysis of variance followed by SNK test (b, p0.0000; e, p = 0.0113; f, p = 0.0000; g, p = 0.0000); n = 3.