OsCASP1 Is Required for Casparian Strip Formation at Endodermal Cells of Rice Roots for Selective Uptake of Mineral Elements

Abstract

In response to diverse environmental conditions, rice (Oryza sativa) roots have developed one Casparian strip (CS) at the exodermis and one CS at the endodermis. Here, we functionally characterized OsCASP1 (Casparian strip domain protein 1) in rice. OsCASP1 was mainly expressed in the root elongation zone, and the protein encoded was first localized to all sides of the plasma membrane of endodermal cells without CS, followed by the middle of the anticlinal side of endodermal cells with CS. Knockout of OsCASP1 resulted in a defect of CS formation at the endodermis and decreased growth under both soil and hydroponic conditions. Mineral analysis showed that the oscasp1 mutants accumulated more Ca, but less Mn, Zn, Fe, Cd, and As in the shoots compared with the wild type. The growth inhibition of the mutants was further aggravated by high Ca in growth medium. The polar localization of the Si transporter Low Si 1 at the distal side of the endodermis was not altered in the mutant, but the protein abundance was decreased, resulting in a substantial reduction in silicon uptake. These results indicated that OsCASP1 is required for CS formation at the endodermis and that the CS in rice plays an important role in root selective uptake of mineral elements, especially Ca and Si.

Figure 1.

Expression Pattern of OsCASP1 in Rice. (A) Relative expression of five OsCASP genes (OsCASP1, OsCASP2, OsCASP3, OsCASP4, and OsCASP5) in the roots. (B) Organ-dependent expression of OsCASP1. Different organs from wild-type rice (cv Nipponbare) were sampled at the heading stage and subjected to RNA extraction and expression determination. (C) Root spatial expression of OsCASP1. RNA was extracted from different segments (0 to 3, 3 to 6, 6 to 9, 9 to 12, and 12 to 15 mm from root apex) of 5-d-old seedlings. Histone H3 was used as an internal standard. Data are means ± sd of three biological replicates.

Figure 2.

Spatial Localization of OsCASP1 and Casparian Strip Formation. (A) to (H) Triple staining for GFP (green), cellulose (blue), and lignin (magenta) was performed in the roots of the transgenic plants carrying the ProOsCASP1-OsCASP1-GFP construct. (E), (F), (G), and (H) are magnified 3D images of the yellow box area in (A), (B), (C), and (D), respectively. (I) to (L) show lignin signal in (E), (F), (G), and (H), respectively. Root cross sections were made at 3 mm (A), 5 mm (B), 15 mm (C), or 30 mm (D) from the apex. Scale bars, 100 μm.

Figure 3.

Observation of Casparian Strip Formation in Wild-Type Rice and Knockout Lines of OsCASP1. (A) to (D) Observation of Casparian strip formation in the wild type (WT) and two mutants. Lignin staining was performed in the roots (20 mm from the apex) of the wild type (A) and (B), oscasp1-1 (C), and oscasp1-2 (D). Magenta shows signal of lignin and blue shows cellulose. (E) to (M) PI penetration test in the roots of the wild type (E), (F), and (K), oscasp1-1 (G), (H), and (L), and oscasp1-2 (I), (J), and (M) at 10 mm (E) to (J) and 20 mm (K) to (M) from the root apex. Eight-day-old seedlings were exposed to PI in the dark for 1 h. (F), (H), and (J) are magnified images of the yellow boxed area in (E), (G), and (I), respectively. en, Endodermis; x, xylem vessel. Casparian strip is indicated by yellow arrowheads. Scale bars, 20 μm (A) to (D), 100 µm (E), (G), (I), (K), (L), and (M).

Figure 4.

Phenotypic Analysis of OsCASP1 Knockout Lines. (A) Phenotype of two independent knockout lines grown in hydroponic solution. Bar, 10 cm. (B) and (C) Fresh weight of the shoots (B) and roots (C). The wild type, oscasp1-1, and oscasp1-2 were grown in a nutrient solution for 34 d. (D) Phenotype of two independent knockout lines grown in soil at the vegetative stage. The wild type, oscasp1-1, and oscasp1-2 were grown under flooded condition for 1 month. Bar, 10 cm. (E) Complementation test. The wild type, oscasp1-1, and two independent complementation lines carrying an OsCASP1 genomic fragment were grown until ripening under flooded condition. Bar, 10 cm. (F) to (I) Comparison of agronomic traits of wild-type (WT) and oscasp1 plants. Tiller number per plant (F), plant height (G), panicle length (H), and grain number per plant (I). Bar, 5 cm. The data are presented as means ± sd (n = 3). Significant difference was determined by Tukey’s test and labeled with different letters (P < 0.05).

Figure 5.

Mineral Elemental Concentration in the Shoots of Wild-Type Rice and Two OsCASP1 Knockout Lines. (A) Concentration of Ca, Sr, and P in the shoots. (B) Concentration of As, Cd, Fe, Ge, Mn, and Zn in the shoots. (C) Concentration of K, Mg, Pb, and Cu in the shoots. Both wild-type rice (WT) and the knockout lines were cultivated in a nutrient solution for 33 d and then transferred to the same solution in the presence of Ge, Sr, Rb, As, and Cd for 1 d before harvest. The data are presented as means ± sd (n = 3). Significant difference was determined by Tukey’s test and labeled with different letters (P < 0.05).

Figure 6.

Effect of Knockout of OsCASP1 on Silicon Uptake, and Expression and Localization of the Silicon Transporter Lsi1 in Rice. (A) and (B) Time-dependent uptake of water (A) and Si (B). (C) Expression of Lsi1 in roots of both the wild type (WT) and oscasp1 mutants. RNA was extracted from different segments of the roots (0 to 6, 6 to 12, and 12 to 18 mm from root apex) of 5-d-old seedlings of the wild type and oscasp1 mutants. Histone H3 was used as an internal standard. Data are means ± sd of three biological replicates. Significant difference was determined by Tukey’s test and labeled with different letters (P < 0.05). (D) to (G) Localization of Lsi1 in the wild type and oscasp1 mutants. Triple staining for Lsi1 (green), cellulose (blue), and lignin (red) was performed in the seminal roots of the wild type (D) and (E), oscasp1-1 (F), and oscasp1-2 (G). (E) Magnified image of the yellow boxed area in (D). Root cross sections were made at 20 mm from the apex. Scale bars, 100 μm.

Figure 7.

Growth of OsCASP1 Knockout Lines at Different Ca Concentrations. (A) to (D) Growth of wild-type rice (WT) and two OsCASP1 knockout lines. Bar, 10 cm. (E) and (F) Dry weight of the shoots and roots. (G) and (H) Ca concentration in the shoots (G) and roots (H). Seedlings (2 weeks old) were grown in a nutrient solution containing 0.18, 1, 5, or 10 mm CaCl₂ for 12 d. (I) Ca concentration in the xylem sap. Xylem sap was collected from the wild type and knockout lines exposed to different Ca concentrations (0.18, 1, 5, and 10 mm) for 6 h. The data are presented as means ± sd (n = 3). Significant difference was determined by Tukey test and labeled with different letters (P < 0.05).