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. 2014 Nov;65(20):6013-21.
doi: 10.1093/jxb/eru340. Epub 2014 Aug 23.

Overexpression of OsHMA3 Enhances Cd Tolerance and Expression of Zn Transporter Genes in Rice

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Free PMC article

Overexpression of OsHMA3 Enhances Cd Tolerance and Expression of Zn Transporter Genes in Rice

Akimasa Sasaki et al. J Exp Bot. .
Free PMC article

Abstract

As a member of the heavy metal ATPase (HMA) family, OsHMA3 is a tonoplast-localized transporter for Cd in the roots of rice (Oryza sativa). Overexpression of OsHMA3 selectively reduces Cd accumulation in the grain. Further characterization in the present study revealed that overexpression of OsHMA3 also enhances the tolerance to toxic Cd. The growth of both the roots and shoots was similar in the absence of Cd between an OsHMA3-overexpressed line and vector control, but the Cd-inhibited growth was significantly alleviated in the OsHMA3-overexpressed line. The overexpressed line showed higher Cd concentration in the roots, but lower Cd concentration in the shoots compared with the wild-type rice and vector control line, indicating that overexpression of OsHMA3 enhanced vacuolar sequestration of Cd in the roots. The Zn concentration in the roots of the OsHMA3-overexpressed line was constantly higher than that of vector control, but the Zn concentration in the shoots was similar between the overexpressed line and vector control. Five transporter genes belonging to the ZIP family were constitutively up-regulated in the OsHMA3-overexpressed line. These results suggest that shoot Zn level was maintained by up-regulating these genes involved in the Zn uptake/translocation. Taken together, overexpression of OsHMA3 is an efficient way to reduce Cd accumulation in the grain and to enhance Cd tolerance in rice.

Keywords: Cd; Oryza sativa; OsHMA3; Zn transporter; overexpression; tolerance..

Figures

Fig. 1.
Fig. 1.
Effect of overexpression of OsHMA3 on Cd tolerance in rice. (A) Phenotype of OsHMA3 overexpressed line (OX), vector control line (VC), and non-transgenic wild-type rice (WT, cv. Nipponbare). (B) Root dry weight of the three lines. (C) Shoot dry weight of the three lines. All lines were cultivated in one-half strength Kimura B solution containing 0, 100, and 1000nM Cd for 22 d. Data are means±SD of three biological replicates. Statistical comparison was performed by one-way ANOVA followed by the Tukey’s test. All data were compared with the wild type, vector control, and overexpression line in each treatment (*P<0.05 and **P<0.01).
Fig. 2.
Fig. 2.
Concentration of Cd and Zn in the roots and shoots of the OsHMA3 overexpressed line. An OsHMA3 overexpressed line (OX), vector control line (VC), and non-transgenic wild-type rice (WT, cv. Nipponbare) were grown in one-half strength Kimura B solution containing 0, 100, and 1000nM Cd for 22 d. The concentration of Cd (A, B) and Zn (C, D) in the roots (A, C) and shoots (B, D) was determined with ICP-MS. Data are means±SD of three biological replicates. Statistical comparison was performed by one-way ANOVA followed by the Tukey’s test. All data were compared with the wild type, vector control and overexpression line in each treatment (*P<0.05 and **P<0.01).
Fig. 3.
Fig. 3.
Concentration of Cu, Fe, and Mn in the roots and shoots. An OsHMA3 overexpressed line (OX), vector control line (VC), and non-transgenic wild-type rice (WT, cv. Nipponbare) were grown in one-half strength Kimura B solution containing 0, 100, and 1000nM Cd for 22 d. The concentration of Cu (A, B), Fe (C, D), and Mn (E, F) in the roots (A, C, E) and shoots (B, D, F) was determined with ICP-MS. Data are means±SD of three biological replicates. Statistical comparison was performed by one-way ANOVA followed by the Tukey’s test. All data were compared with the wild type, vector control and overexpression line in each treatment (*P<0.05 and **P<0.01).
Fig. 4.
Fig. 4.
Time-dependent change of Cd and Zn in the root cell saps. Seedlings of an OsHMA3 overexpressed line (OX) and non-transgenic wild-type rice (WT, cv. Nipponbare) were exposed to 500nM Cd for different times. Concentration of Cd (A) and Zn (B) in the root cell sap was determined with ICP-MS. Data are means±SD of four biological replicates. Asterisks indicate significant difference from WT at *P<0.05 and **P<0.01 by Student’s t-test.
Fig. 5.
Fig. 5.
Time-dependent accumulation of 67Zn in the roots. Seedlings of an OsHMA3 overexpressed line (OX) and non-transgenic wild-type rice (WT, cv. Nipponbare) were exposed to 500nM 67Zn for different times. The concentration of newly accumulated Zn (∆Zn) was calculated by 66Zn/67Zn ratio determined with isotope mode of ICP-MS. Data are means±SD of four biological replicates. Asterisks indicate significant difference from WT at **P<0.01 by Student’s t-test.
Fig. 6.
Fig. 6.
Effect of OsHMA3 overexpression on uptake of Ni, Pb, and Co. Seedlings of an OsHMA3 overexpressed line (OX) and non-transgenic wild-type rice (WT, cv. Nipponbare) were exposed to 500nM Ni, Pb, or Co for 24h. The concentration of metals was determined using ICP-MS. Data are means±SD of four biological replicates. Asterisks indicate significant difference from WT at *P<0.05 and **P<0.01 by Student’s t-test.
Fig. 7.
Fig. 7.
Expression of ZIP genes in the roots. Seedlings of an OsHMA3 overexpressed line (OX) and vector control (VC) were exposed to 0 or 200nM Cd for 24h. The expression of ten ZIP genes in the roots was determined by quantitative real-time PCR. Histone H3 and Actin was used as internal standards. Expression relative to VC (–Cd) is shown. Statistical comparison was performed by one-way ANOVA followed by the Tukey’s test. Data are means±SD of three biological replicates. Different letters indicate significant difference at P<0.05.

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