Identification of a rice metal tolerance protein OsMTP11 as a manganese transporter

Abstract

Metal tolerance proteins (MTPs) are a gene family of cation efflux transporters that occur widely in plants and might serve an essential role in metal homeostasis and tolerance. Our research describes the identification, characterization, and localization of OsMTP11, a member of the MTP family from rice. OsMTP11 was expressed constitutively and universally in different tissues in rice plant. Heterologous expression in yeast showed that OsMTP11 complemented the hypersensitivity of mutant strains to Mn, and also complemented yeast mutants to other metals, including Co and Ni. Real time RT-PCR analysis demonstrated OsMTP11 expression was substantially enhanced following 4 h under Cd, Zn, Ni, and Mn treatments, suggesting possible roles of OsMTP11 involvement in heavy metal stress responses. Promoter analysis by transgenic assays with GUS as a reporter gene and mRNA in situ hybridization experiments showed that OsMTP11 was expressed specifically in conducting tissues in rice. DNA methylation assays of genomic DNA in rice treated with Cd, Zn, Ni, and Mn revealed that decreased DNA methylation levels were present in the OsMTP11 promoter region, which was consistent with OsMTP11 induced-expression patterns resulting from heavy metal stress. This result suggested that DNA methylation is one of major factors regulating expression of OsMTP11 through epigenetic mechanisms. OsMTP11 fused to green fluorescent protein (GFP) localized to the entire onion epidermal cell cytoplasm, while vacuolar membrane exhibited increased GFP signals, consistent with an OsMTP11 function in cation sequestration. Our results indicated that OsMTP11 might play vital roles in Mn and other heavy metal transportation in rice.

Fig 1. Bioinformatics analyses of OsMTP11 nucleotide and amino acid sequences.

A. OsMTP11 gene structure analysis in the Rice Genome Annotation Database (http://rice.plantbiology.msu.edu/). B. Phylogenetic tree of the MTP family from rice and Arabidopsis. The tree was constructed using MEGA 4.0.2 by the neighbor-joining method. Arabidopsis MTP amino acid sequences were obtained from www.tigr.org: AtMTP1, At2g46800; AtMTP2, At3g61940; AtMTP3, At3g58810; AtMTP4, At2g29410; AtMTP5, At3g12100; AtMTP6, At2g47830; AtMTP7, At1g51610; AtMTP8, At3g58060; AtMTP9, At1g79520; AtMTP10, At1g16310; AtMTP11, At2g39450; AtMTP12, At2g04620. Rice MTP amino acid sequences were downloaded from http://rice.plantbiology.msu.edu/. OsMTP11, Os01g62070; OsMTP1, Os05g03780; OsMTP5, Os02g58580; OsMTP6, Os03g22550; OsMTP7, Os04g23180; OsMTP8, Os02g53490; OsMTP8.1, Os03g12580; OsMTP9, Os01g03914; OsMTP11, Os01g62070; OsMTP11.1, Os05g38670; OsMTP12, Os08g32680. C. Amino acid alignment of OsMTP11, AtMTP11 [11] and ShMTP1 (AY181256) [12]. Amino acid sequences of four predicted transmembrane (TM) segments are boxed. Amino acid residues with dark shading indicate conserved sequences, and residues with light gray shading indicate those conserved in two protein sequences. D. The predicted transmembrane helices of OsMTP11. The transmembrane domains were estimated using TMHMM2: www.cbs.dtu.dk/services/TMHMM/. The peaks show the predicted transmembrane (TM) regions of proteins. These data indicate that OsMTP11 has four obvious TM regions.

Fig 2. Expression pattern of OsMTP11 by real time RT-PCR.

A. Real time RT-PCR results of OsMTP11 expression in wild-type rice plants (Nipponbare) from different tissues or organs. The amplification of the rice OsUBQ5 (AK061988) gene was used as a control to normalize the transcript level of OsMTP11. B. Expression analysis of OsMTP11 under different heavy metal stresses (Mn, Cd, Zn and Ni) by real time RT-PCR. The expression of OsMTP11 is increased in rice roots and shoots treated with 0.5 mM CdCl₂, 5 mM Zn(NO₃)₂, 1 mM NiCl₂, 2 mM MnSO₄, 300 mM NaCl and 100 μM methyl viologen (MV) for different time periods. C. Expression analysis of OsMTP11 under 300 mM NaCl and 100 μM methylviologen (MV) by real time RT-PCR.

Fig 3. Complementation of yeast mutants on selective medium.

A. OsMTP11 and AtMTP11 genes complement the Mn-hypersensitive phenotype of a pmr1Δ yeast mutant on selective medium (SDG medium) without or with 1 mM Mn. B. OsMTP11 complemented the Co-hypersensitive phenotype of the cot1Δ yeast mutant and Ni-hypersensitive phenotype of the smf1Δ yeast mutant, but could not complement the Zn-hypersensitive phenotype of the zrc1Δ yeast mutant, the Cd-hypersensitive phenotype of the ycf1Δ yeast mutant and the Cu-hypersensitive phenotype of the cup2Δ yeast mutant. C. OsMTP11 is incapable of complementing the yeast vacuolar acidification mutants vph2Δ and vma8Δ.

Fig 4. Effect of OsMTP11 expression in wild yeast BY4741 and yeast mutants for heavy metal tolerance and cation content.

A. Yeast mutant (pmr1Δ) transformed with the empty plasmid vector (pYES260) or MTP genes in pYES260 (OsMTP11 and AtMTP11) were grown in either SDG liquid medium or SDG liquid medium supplemented with 0.1 mM Mn²⁺. Culture optical density at 600 nm (OD₆₀₀) was determined at 12 h intervals. B. Yeast mutants (smf1Δ and cot1Δ) transformed with the empty plasmid vector (pYES260) or the OsMTP11 gene in pYES260 were grown in either SDG liquid medium or SDG liquid medium supplemented with 0.2 mM Ni²⁺ (smf1Δ), 3.5 mM Mn²⁺ (smf1Δ) and 0.2 mM Co²⁺ (cot1Δ). Culture optical density at 600 nm (OD₆₀₀) was determined at 12 h intervals. C. Wild type yeast BY4741 transformed with the empty plasmid vector (pYES260) or the OsMTP11 gene in pYES260 was grown in either SDG liquid medium or SDG liquid medium supplemented with 3.5 mM Mn²⁺, 0.2 mM Co²⁺ and 0.15 mM Ni²⁺. Culture optical density at 600 nm (OD₆₀₀) was determined at 12 h intervals. D. OsMTP11 expression affects heavy metal content in yeast. Wild type yeast BY4741 transformed with the empty plasmid vector (pYES260) or the OsMTP11 gene in pYES260 was grown in SDG liquid medium supplemented with 3.5 mM Mn²⁺, 0.2 mM Co²⁺ and 0.15 mM Ni²⁺. The yeast was harvested at an OD₆₀₀ value as 1.5~1.8, then the heavy metal was measured with ICP-AAS. Results represent means ± standard error of three biological replicates. Single asterisk symbols (*) indicate significant differences between control (empty vector pYES260) and experimental samples (OsMTP11- pYES260) at an individual detection (0.01 < P < 0.05); Double asterisk symbols (**) indicate significant differences between control (empty vector pYES260) and experimental samples (OsMTP11- pYES260) at an individual detection (P < 0.01).

Fig 5. DNA methylation analysis of the OsMTP11 promoter region.

A. Schematic distribution of CpG sites and CpG islands in the OsMTP11 promoter region. Blue area indicates CpG islands. The scale bar indicates 100 bp. Red bar indicates the region analyzed by bisulfate sequencing. B. Comparison of percentages of cytosine methylation of three different types (CpG, CpHpG, and CpHpH) in rice seedling roots between control and under heavy metal stress (0.5 mM CdCl₂, 5 mM ZnSO_4, 2 mM MnSO_4, 1 mM NiCl₂) or salt treatment (300 mM NaCl) for 72 hours.

Fig 6. Transient expression of OsMTP11::EGFP in onion epidermal cells.

A. Localization of EGFP control. B. Bright-field and fluorescence merged image of A. C. Localization of OsMTP11::EGFP. D. Bright-field and fluorescence merged image of C.

Fig 7. mRNA in situ hybridization of OsMTP11 in rice leaves.

Blue or purple precipitates indicate positive OsMTP11 mRNA signals. A. Transverse sections of a mature leaf. B. Transverse sections of a young leaf bud. C. Longitudinal sections of a seedling leaf bud. D. Control with the transverse sections of leaf bud.

Fig 8. OsMTP11 native promoter-drived GUS staining in rice leaves and roots.

Blue precipitates indicate positive GUS signals. A. Leaf with GUS staining in vein tissue. B. Root tip with GUS staining in conducting tissue. C.Transverse sections of A. D. Transverse sections of B.

Fig 9. The OsMTP11 putative promoter region (-2,250 bp) sequence.

The transcription start site is denoted +1, and the putative start codon is underlined. Diagram of the OsMTP11 promoter region using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) showed the presence of a number of potential cis-acting elements that respond to environmental signals. MRE, metal-response element [34, 35]; ABRE, abscisic acid-response element [36]; I-box, light-response element [37]; BS1EGCCR, "BS1 (binding site 1)" found in CCR gene promoter, which is a cis-element required for vascular expression of the cinnamoyl CoA reductase gene in E. gunnii [38]. MREs include MRE1: 5’-TGCRCNC-3’ (R = A or G; N = any residue) [34] and MRE2: 5’-HTHNNGCTGD-3’ (D = A, G, or T; H = A, C, or T; N = any residue) [35].