Role of polar localization of the silicon transporter OsLsi1 in metalloid uptake by rice roots

Abstract

Low silicon (Si) rice 1 (OsLsi1) is a key transporter mediating Si uptake in rice (Oryza sativa). It is polarly localized at the distal side of the root exodermis and endodermis. Although OsLsi1 is also permeable to other metalloids, such as boron (B), germanium (Ge), arsenic (As), antimony (Sb), and selenium (Se), the role of its polar localization in the uptake of these metalloids remains unclear. In this study, we investigated the role of OsLsi1 polar localization in metalloid uptake by examining transgenic rice plants expressing polarly or nonpolarly localized OsLsi1 variants. Loss of OsLsi1 polar localization resulted in decreased accumulation of Ge, B, and As in shoots but increased Sb accumulation, while Se accumulation remained unaffected under normal conditions. Experiments with varying B concentrations revealed that B uptake is significantly lower at low B concentrations (0.3 to 3 μm) but higher at high B concentrations (300 μm) in plants expressing nonpolarly localized OsLsi1, despite the similar B permeability of both OsLsi1 variants in Xenopus oocytes and their comparable protein abundance in roots. Additionally, the loss of OsLsi1 polarity did not affect the abundance, localization, or high B-induced degradation of the borate transporter 1 (OsBOR1), an efflux transporter that cooperates with OsLsi1 for B uptake. Taken together, our findings demonstrate that the polar localization of OsLsi1 plays a critical role in regulating metalloid uptake, depending on the presence or absence of efflux transporters cooperating with OsLsi1.

Figure 1.

Comparison of shoot metalloid accumulation in plants harboring polar and nonpolar OsLsi1. A to E) Concentrations of B A), As B), Ge C), Sb D), and Se E) in shoots of plants harboring polar and nonpolar OsLsi1. Seedlings (6-d-old) of WT (WT rice), lsi1-3, 2 independent Flag-OsLsi1 transgenic lines, and 3 independent Flag-OsLsi1^{I18A, I285A} were grown in a hydroponic solution containing 3 μm B for 15 d A, B) or 12 d C to E). Before harvest, the plants were exposed to a solution containing 2 μm of Ge, As, Se, or Sb in the presence of 3 μm B for 24 h. Concentration of metalloids was determined by ICP-MS. Data are means ± Sd (n = 3 to 4). Different letters indicate significant differences at P < 0.05 by Tukey–Kramer's test. DW, dry weight.

Figure 2.

Effect of OsLsi1 polar localization on B uptake at different B concentrations. A to C) B concentration in the shoots A) and roots B) and B uptake C). Seedlings (6-d-old) of WT (WT rice), lsi1-3, 2 independent Flag-OsLsi1 transgenic lines, and 3 independent Flag-OsLsi1^{I18A, I285A} lines were grown in solutions containing 0.3, 3, 30, or 300 μm B for 21 d. Concentration of B in the roots and shoots was determined by ICP-MS. Uptake was calculated by (shoot B content + root B content)/root dry weight. Data are means ± Sd (n = 4). Different letters indicate a significant difference at P < 0.05 by Tukey–Kramer's test. DW, dry weight.

Figure 3.

Effect of OsLsi1 polar localization on short-term ¹⁰B uptake at different ¹⁰B concentrations. A, B)  ¹⁰B concentration in the shoots A) and roots B) and ¹⁰B uptake C). Seedlings (6-d-old) of WT (WT rice), lsi1-3, 2 independent Flag-OsLsi1 transgenic lines, and 3 independent Flag-OsLsi1^{I18A, I285A} lines were precultured in solutions containing 0.3, 3, or 300 μm  ¹¹B for 33 d. Before harvest, the plants were exposed to a solution containing 0.3, 3, or 300 μm  ¹⁰B for 24 h. Concentration of ¹⁰B in the roots and shoots was determined by ICP-MS using an isotope model. ¹⁰B uptake was calculated by (shoot ¹⁰B content + root ¹⁰B content)/root dry weight. Data are means ± Sd (n = 4). Different letters indicate significant differences at P < 0.05 by Tukey–Kramer's test. DW, dry weight.

Figure 4.

Comparison of expression level of OsLsi1 and OsBOR1 in roots of plants expressing polar and nonpolar OsLsi1 at low and high B concentrations. A, B) Expression level of OsLsi1  A) and OsBOR1  B) in the roots. Seedlings (18-d-old) of WT (WT rice), lsi1-3, 2 independent Flag-OsLsi1 transgenic lines, and 3 independent Flag-OsLsi1^{I18A, I285A} lines were grown in solutions containing 0.3 or 300 μm B. After 3 d, the roots were sampled for RNA extraction. Expression of OsLsi1 and OsBOR1 was determined by reverse transcription quantitative PCR. HistoneH3 and Ubiquitin genes were used as internal controls. Expressions relative to WT at 0.3 μm B are shown. Data are means ± Sd (n = 4). Different letters indicate significant differences at P < 0.05 by Tukey–Kramer's test.

Figure 5.

Response of OsLsi1 and OsBOR1 proteins in the roots of plants expressing polar and nonpolar OsLsi1 to different B concentrations. A to C) Western blotting of Flag-OsLsi1 variants A), OsBOR1 B), and H⁺-ATPase C). Seedlings (22-d-old) of transgenic plants carrying Flag-OsLsi1 (line67) and Flag-OsLsi1^{I18A, I285A} (line8) were exposed to a solution containing 0.3, 3, 30, and 300 μm B for 3 d. The knockout mutant of OsBOR1 (bor1-1) treated with 0.3 μm B for 3 d was used as a negative control of Flag and OsBOR1 antibodies. The same amount of protein (7 μg) of microsome fraction was analyzed by SDS-PAGE and visualized by western blot with antibodies against Flag, OsBOR1, and H⁺-ATPase (as an internal standard). MM indicates molecular marker.

Figure 6.

Cellular and subcellular localization of OsLsi1 variants and OsBOR1 at different B concentrations in roots of plants harboring polar and nonpolar OsLsi1. A to C) Double immunostaining of Flag-OsLsi1 variants (green color, upper panel) and OsBOR1 (magenta color, lower panel) in transgenic plants harboring polar OsLsi1 A), nonpolar OsLsi1 B), and knockout mutant of OsBOR1 (bor1-1) C), as a negative control, at different B concentrations from 0.3 to 300 μm B. D to I) Enlarged images of Flag-OsLsi1 variants (green color, upper panel) and OsBOR1 (magenta color, lower panel) at exodermis D to F) and endodermis G to I) of the roots. Seedlings (28-d-old) of transgenic lines carrying Flag-OsLsi1 (line67) and Flag-OsLsi1^{I18A, I285A} (line8) were exposed to a solution containing 0.3, 3, 30, and 300 μm B for 3 d. The bor1-1 treated with 0.3 μm B for 3 d was used as a negative control of Flag and OsBOR1 antibodies. Cross-sections of the mature region (10 to 20 mm from the root tip) of the crown root were prepared for immunostaining with Flag and OsBOR1 antibodies, which signals were merged with autofluorescence of the cell wall, indicated by blue color A to C). Bars indicate 50 A to C) or 10 μm D to J). Whole root and enlarged images were taken under the same conditions for comparison, respectively. J, K) Quantified signal intensity of polar and nonpolar OsLsi1 variants J) and OsBOR1 K). Signal intensities from Flag and OsBOR1 antibodies of the root images were quantified by LAS AF Lite software J, K). The signal of bor1-1 was subtracted as a background, and signal intensity relative to 0.3 μm B condition was shown J, K). Data are means ± Sd from 10 independent slices. Different letters indicate significant differences at P < 0.05 by Tukey–Kramer's test.

Figure 7.

Schematic presentation on the role of OsLsi1 polar localization in metalloid uptake in rice. A, B) Role of polar localization of OsLsi1 in metalloid uptake in the presence of efflux transporters. When OsLsi1 is polarly localized at the distal side of the exodermis and endodermis A), it forms a directional transport pathway with efflux transporters (OsBOR1 or OsLsi2), facilitating the uptake of metalloids, including B, Si, Ge, and As. However, when OsLsi1 loses its polar localization B), these metalloids effluxed by the efflux transporters to the apoplastic space are retaken up by OsLsi1 localized at the proximal side, decreasing the efficiency of the uptake. C, D) Role of polar localization of OsLsi1 in metalloid uptake in the absence of efflux transporters. Due to lack of efflux transporter (Sb) or downregulation of OsBOR1 (B at high concentrations), these metalloids taken up by polar OsLsi1 are retained in the exodermal cells, resulting in less root-to-shoot translocation of these metalloids C). However, nonpolar OsLsi1 at the proximal side facilitates the flow of Sb and B toward the stele side D), resulting in increased uptake of these metalloids. Green indicates OsLsi1 localization, while magenta indicates localization of efflux transporters, OsBOR1 and OsBOR2. Blue arrows indicate the flow of metalloids from the soil to the steel, while orange arrows indicate the reuptake of metalloid. CS indicates Casparian strips.