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Expression of rice MATE family transporter OsMATE2 modulates arsenic accumulation in tobacco and rice

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The OsMATE2 upon constitutive expression in tobacco decreases root-to-shoot As transfer coefficient and its endosperm-specific silencing in rice reduces grain As content, broadening the role of MATE proteins in planta.

Abstract

Rice (Oryza sativa) is capable of accumulating significant amount of arsenic (As) in grains, causing serious health hazard for rice consuming population. The multidrug and toxic compound extrusion (MATE) protein family comprises a large group of secondary transporters present universally in living organisms, and transports metabolites and/or xenobiotic compounds. OsMATE2, one of the MATE family members of rice was found to be transcriptionally up-regulated (sixfolds) in the developing seeds during As stress, and showed positive correlation with the As content in mature grains. Therefore, to understand the role of OsMATE2 in As accumulation, constitutive expression in tobacco was carried out. Transgenic tobacco plants exhibited decreased root-to-shoot As transfer coefficient (33.3–39.6%) along with augmented As sensitivity by increasing oxidative stress compared to untransformed control plants, indicating the involvement of OsMATE2 in As accumulation. Consequently, RNAi strategy was utilized for endosperm-specific silencing of endogenous OsMATE2 to mitigate As accumulation in rice grains. Transgenic rice lines demonstrated significant reduction of both OsMATE2 transcript (~ 38–87%) and grain As content (36.9–47.8%) compared to the control plants without undesirable effects on agronomical traits. Together, the present findings indicate the connection of OsMATE2 in As accumulation, and could expand the functional role of MATE proteins in planta.

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Acknowledgements

We sincerely thank late Prof. Soumitra K. Sen and Dr. Asitava Basu for their cooperation and help. The authors also acknowledge the help received from Dr. Tirthartha Chattopadhyay and Dr. Sheuli Roy during the initial phase of the study. We also acknowledge the technical help received from Mr. Sona Dogra, Mrs. Gayatri Aditya, Mr. Manoj Aditya and Mr. Nitai Giri. This work was supported by the grants from DBT, Govt. of India (BT/PR12907/AGR/36/639/2009), and the IIT Kharagpur Food Security Project (F. No. 4-25/2013-TS-1).

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  1. Surajit Bhattacharya

    Present address: Amity Institute of Biotechnology, Amity University, Rajarhat, Newtown, Kolkata, 700135, India

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Natasha Das, Surajit Bhattacharya & Mrinal K. Maiti

  2. Department of Genetics, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, 741252, India

    Somnath Bhattacharyya

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  1. Natasha Das
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Contributions

Experimental designs and analyses of results were carried out by ND and MKM. ND conducted the experiments. SB1 helped ND in conducting few experiments and preparing the manuscript. MKM and SB2 conceived the original research plan. MKM made the necessary corrections in the manuscript and supervised the research work.

Corresponding author

Correspondence to Mrinal K. Maiti.

Electronic supplementary material

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11103_2018_766_MOESM1_ESM.tif

Supplementary figure S1. In silico analysis of the OsMATE2 polypeptide derived from the newly cloned OsMATE2 CDS of indica rice cultivar IR64. (A) Dompred server (http://bioinf.cs.ucl.ac.uk/psipred/?dompred=1) based prediction of the secondary structure of the OsMATE2 polypeptide. (B) MEMSAT-SVM server (http://bioinf.cs.ucl.ac.uk/psipred/?memsatsvm=1) mediated protein topology prediction using support vector machine-based (SVM) structures, highlighting the presence of 12 transmembrane helices in the OsMATE2 polypeptide. (TIF 1415 KB)

11103_2018_766_MOESM2_ESM.tif

Supplementary figure S2. Multiple sequence alignment of deduced amino acid sequences of OsMATE2 CDS with selected characterized MATE transporter orthologs, which are NorM of Vibrio cholerae, NorM of V. parahaemolyticus, ALF5 of A. thaliana, Jat1 of N. tabacum. The red boxes indicate the presence of the two amino acids E279 and D397 critical for cation binding in the OsMATE2 protein for the transporter activity. (TIF 3371 KB)

11103_2018_766_MOESM3_ESM.tif

Supplementary figure S3. Phylogenetic relationship of OsMATE2 using a few functionally characterized and non-characterized members of the MATE family proteins of plant kingdom including the representative members of both monocots and dicots. The unrooted phylogenetic tree was obtained on the basis of a Clustal Omega protein sequence alignment using the protein maximum likelihood (PhyML) algorithm. Analysis using 500 bootstrap replicates was performed. Scale bar indicates expected changes per site. (TIF 4782 KB)

11103_2018_766_MOESM4_ESM.tif

Supplementary figure S4. Homology modelling of OsMATE2 and in silico analysis. (A) The predicted model of OsMATE2 based on the crystal structure of AtDTX14 of A. thaliana (PDB ID 5Y50) showing the bi-lobed structure composed of N-lobe (TM 1-6) and C-lobe (TM 7-12) based on the arrangement of the 12 TM domains, forming a V-shaped structure open toward the extracellular side. (B) The position of the salt bridge in the predicted model of OSMATE2. (C) Interaction between Glu 103 (TM2) and Arg 408 (TM10) residues in the salt bridge, which may play critical role in transport activity of the protein by regulating the closing of the internal gate. (D) Position of the external gate in the predicted model of OsMATE2. (E) The external gate is composed of the residue pairs Asp 153 & Asn 295, and Ala 81 & Ser 304 along with the Gly 291, which together may contribute to the inward open structure of OsMATE2. (TIF 13548 KB)

11103_2018_766_MOESM5_ESM.tif

Supplementary figure S5. (A) EtBr stained agarose gel (0.8%) electrophoresis for verification of the pCAM::2xCaMV35S-OsMATE2-NOS recombinant binary plasmid by different restriction enzyme digestions. Lanes: 200 bp DNA ladder as molecular weight marker (lane 1), recombinant plasmid undigested (lane 2), and digested with BamHI+HindIII (lane 3), BamHI (lane 4), BamHI+KpnI (lane 5), KpnI (lane 6), SacI (lane 7) and XhoI (lane 8). (B) Representative Southern blot showing the integration pattern of the OsMATE2 transgene in T1 transgenic tobacco lines. Arrowheads indicate the position of the HindIII digested lambda DNA as a molecular weight marker. (C) EtBr stained agarose gel (0.8%) electrophoresis for verification of the pCAM::GluC-hpOsMATE2-NOS recombinant binary plasmid by different restriction enzyme digestions. Lanes: pUC18 DNA digested with HinfI as molecular weight marker (M), recombinant plasmid digested with HindIII (lane 1), BamHI (lane 2), BamHI+ HindIII (lane 3), PstI (lane 4) and KpnI (lane 5). (D) Representative Southern blot showing the integration pattern of the hp-OsMATE2 transgene in T2 transgenic rice lines. Lanes: untransformed control (Con), t independent T2 transgenic lines (hpL1- hpL6). Arrowheads indicate the position of HindIII digested lambda DNA as molecular weight marker. (TIF 10792 KB)

11103_2018_766_MOESM6_ESM.tiff

Supplementary figure S6. The universal expression pattern of rice MATE family genes and response of OsMATE2 in As stress of rice. (A) Metaexpression analysis of the MATE family genes in different anatomical tissues/organs or developmental stages of rice plant based on a large collection of Affymetrix microarray data (http://www.ricearray.org/). (B) Expression pattern of OsMATE2 (LOC_Os05g48040) in different anatomical tissues and in various developmental stages of rice plant. (C) Expression pattern of OsMATE2 (LOC_Os05g48040) in response to As treatment in the rice varieties Bala and Azucena, based on publicly available data (GSE4471, Norton et al. 2008). The numbers below the heat map indicate the log2 normalized intensity of the microarray data. (TIFF 2006 KB)

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Das, N., Bhattacharya, S., Bhattacharyya, S. et al. Expression of rice MATE family transporter OsMATE2 modulates arsenic accumulation in tobacco and rice. Plant Mol Biol 98, 101–120 (2018). https://doi.org/10.1007/s11103-018-0766-1

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