Functional characterization of BjCET3 and BjCET4, two new cation-efflux transporters from Brassica juncea L

Abstract

Brassica juncea is promising for metal phytoremediation, but little is known about the functional role of most metal transporters in this plant. The functional characterization of two B. juncea cation-efflux family proteins BjCET3 and BjCET4 is reported here. The two proteins are closely related to each other in amino acid sequence, and are members of Group III of the cation-efflux transporters. Heterologous expression of BjCET3 and BjCET4 in yeast confirmed their functions in exporting Zn, and possibly Cd, Co, and Ni. Yeast transformed with BjCET4 showed higher metal resistance than did BjCET3 transformed. The two BjCET-GFP fusion proteins were localized to the plasma membrane in the roots when expressed in tobacco, and significantly enhanced the plants' Cd tolerance ability. Under Cd stress, tobacco plants transformed with BjCET3 accumulated significant amounts of Cd in shoots, while maintaining similar shoot biomass production with vector-control subjects. Transformed BjCET4 tobacco plants showed significantly enhanced shoot biomass production with markedly decreased shoot Cd content. The two transporter genes have a lower basal transcript expression in B. juncea seedling tissues when grown in normal conditions than under metal-stress, however, their transcripts levels could be substantially increased by Zn, Cd, NaCl or PEG, suggesting that BjCET3 and BjCET4 may play roles in several stress conditions, roles which appear to be different from those of previous characterized cation-efflux transporters, for example, AtMTP1, BjCET2, and BjMTP1.

Fig. 1.

BjCET sequence analysis. (A) Alignments of predicted amino acid sequences for BjCET1, BjCET2, BjCET3, BjCET4, and BjMTP1 from B. juncea, TgMTP1 from T. goesingense and AtMTP1 from A. thaliana. Sequence alignment was performed by CLUSTALW. The following protein features have been highlighted: the six transmembrane domains (TMs) identified by TMHMM (version 2.0) (underlined in dark green); the CDF signature sequences (yellow); the histine-rich loop region (dashed dark green line); the highly conserved aspartate (filled inverted triangles)in domain II and V (Bloss et al., 2002); the C-terminal LZ motif (red) (Blaudez et al., 2003); and the C-terminal putative Zn binding site HD(E)XHXWXL(I)TX₈H (dark green) (Dräger et al., 2004). (B) Phylogenetic analysis of the identified CE proetins from B. juncea. The Neighbor–Joining phylogenetic tree was constructed using MEGA4.1 after CLUSTALW alignment of the full-length amino acid sequences.

Fig. 2.

Phylogenetic analysis of the CE proteins from B. juncea and other genera. The Neighbor–Joining phylogenetic tree based on the protein alignment was created with MEGA 4.1. The sequences were aligned with Clustal X software, and the scale bar indicates an evolutionary distance of 0.05 amino acid substitution per site. The number at the nodes show bootstrap values (>800) after 1000 replicates. Accession numbers and species names of each gene are shown in Supplementary Table S2 at JXB online.

Fig. 3.

sqRT-PCR analysis of steady-state levels of BjCET3 and BjCET4 transcripts and their regulation by Zn, Cd, NaCl, and PEG in root, stem, and shoot tissues of B. juncea. (A) BjCET3 and BjCET4 mRNA expression level in tissues of the 5–6 leaf stage of B. juncea seedlings growing in normal conditions; (B) BjCET3 and BjCET4 expression level in tissues of the 5–6 leaf stage of B. juncea seedlings after 12 h treatment with 200 μM CdCl₂, 1000 μM ZnCl₂, 250 mM NaCl, and 20% w/v PEG6000, respectively; (C) BjCET3 and BjCET4 expression level in tissues of the 5–6 leaf stage of B. juncea seedlings after 4, 8, 12 , and 24 h treatment with 200 μM CdCl₂ and 250 mM NaCl, respectively. 25 cycles were used for all PCR reactions, which were tested in the linear amplification range for each target gene.

Fig. 4.

Quantification of metal tolerance of recombinant yeast. (A–C) Quantification of Cd, Zn and Co tolerance of zrc1&cot1 mutant strains (YK44) expressing BjCET3 and BjCET4, respectively. (D) Quantification of Ni tolerance of cot1 mutant strains (YK40) expressing BjCET3 and BjCET4. (E, F) Quantification of Zn, Cd tolerance of zrc1 mutant strains (YK41) expressing BjCET3 and BjCET4. Metal tolerance was quantified by using the assay system described in Persans et al. (2001). Data are means ±SD of three biological repeats.

Fig. 5.

ICP-MS assay of Zn content in zrc1 yeast cells. Cultured yeast cells (OD₆₀₀=0.4) were incubated with 125 μM ZnCl₂ for 4 h before collection. Then yeast cells were washed with 20 mM EDTA and sterilized deionized water to remove the medium Zn and yeast cell wall unspecific bound Zn. The Zn content inside yeast cells was determined by ICP-MS. A significantly reduced Zn accumulation was detected in BjCET3 (P <0.001) and BjCET4 (P <0.01) expressing yeast compared to control (empty vector transformed) yeast. Comparison between different genotypes was made using a one-way ANOVA test to our endpoints data, followed by a Tukey test when data were statistically different (P <0.05) (Zar, 1996). Values are average of each genotype (arithmetic mean ±SD of n=3 independent experiments).

Fig. 6.

Verifying BjCET3 and BjCET4 expression in transgenic tobaccos and their subcellular localization in roots. (A) Structures of plasmid pBI121-BjCET-GFP. nos-pro, Nopaline synthase promoter; Npt II, neomycin phosphotransferase; nos-ter, nopaline synthase terminator; CaMV 35S Pro, CaMV 35S promoter. (B) RT-PCR analysis of BjCET3 and BjCET4 transcript level in the leaves of transgenic plants. PCR products are their fused GFP cDNA fragments. Actin was used as the control. (C) Subcellular localization of BjCET proteins in root cells of transgenic tobaccos. C1–C3, images of GFP fluorescence in transgenic tobacco root cells in normal growing condition; C4–C6, images of GFP fluorescence in transgenic tobacco root cells treated with saturated sucrose solution for 5–10 min; C7, C8, images of GFP and DAPI (arrow) fluorescence in BjCET3 and BjCET4 transgenic tobacco root cells. Before analysis, root samples were treated with 2 μg ml⁻¹ DAPI for 15 min. C1 and C4 represent pBI121-GFP transgenic tobacco tissues, C2, C5, and C7 represent pBI121-BjCET3-GFP transgenic tobacco tissues, and C3, C6, and C8 represent pBI121-BjCET4-GFP transgenic tobacco tissues. Fluorescence of GFP and DAPI was observed by using confocal laser scanning microscopy under 485 nm and 364 nm, respectively.

Fig. 7.

Overexpression of BjCET protein confers Cd tolerance to tobacco leaf explants. (A) Showing an example of plantlets regenerated from 10 leaf explants of each genotype cultured in medium with or without 200 μM CdCl_2. (B) Quantitative analysis of numbers of regenerated seedlings from each genotype. Comparison between different genotypes upon Cd exposures was made using a one-way ANOVA test to our endpoints data, followed by a Tukey test when data were statistically different (P <0.05) (Zar, 1996). Values are average number of each genotype (arithmetic mean ±SD of n=3 independent experiments). Different letters indicate significant differences between the groups. Control, pBI121-GFP vector transgenic tobacco; BjCET3, pBI121-BjCET3-GFP transgenic tobacco; BjCET4, pBI121-BjCET4-GFP transgenic tobacco. (This figure is available in colour at JXB online.)

Fig. 8.

Over-expression of BiCET3 and BjCET4 enhanced Cd tolerance of transgenic tobaccos. The photographs are representative of the performance of BiCET3 and BjCET4 transgenic tobaccos on days 15 and 50 after planting on MT₁ medium with or without the addition of 200 μM CdCl_2. Control, pBI121-GFP vector transgenic tobacco; BjCET3, pBI121-BjCET3-GFP transgenic tobacco; BjCET4, pBI121-BjCET4-GFP transgenic tobacco. (This figure is available in colour at JXB online.)

Fig. 9.

Cd tolerance and accumulation in roots and shoots of BjCET transgenic plants exposed to CdCl₂. (A–C) Quantitative analysis of root length (A), fresh root weight (B), and fresh shoot weight (C) of different genotype plantlets exposed to 200 μM CdCl₂ for 30 d. (D, E) Root and shoot Cd content (μg mg⁻¹ dry weight) of different genotype plantlets exposed to 200 μM CdCl₂ for 30 d, respectively. (F) Total Cd accumulated in shoot of per plant for each genotype. The statistical analysis method used was the same as Fig. 7. Values are average number of each genotype (arithmetic mean ±SD of n=5–7). Different letters indicate significant differences between the groups.