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Plant Molecular Biology

, Volume 94, Issue 1–2, pp 167–183 | Cite as

Identification of alternatively spliced transcripts of rice phytochelatin synthase 2 gene OsPCS2 involved in mitigation of cadmium and arsenic stresses

  • Natasha Das
  • Surajit Bhattacharya
  • Somnath Bhattacharyya
  • Mrinal K. MaitiEmail author
Article

Abstract

Key message

The OsPCS2 exhibits root- and shoot-specific differential ratios of alternatively spliced transcripts in indica rice under Cd stress, and plays role in Cd and As stress tolerance and accumulation.

Abstract

Enzymatic activity of phytochelatin synthase (PCS) in plant produces phytochelatins, which help in sequestration of heavy metal(loid)s inside the cell vacuole to alleviate toxicity. Here we report that among the two PCS genes—OsPCS1 and OsPCS2 in indica rice (Oryza sativa) cultivar, the OsPCS2 produces an alternatively spliced OsPCS2b transcript that bears the unusual premature termination codon besides the canonically spliced OsPCS2a transcript. Root- and shoot-specific differential ratios of alternatively spliced OsPCS2a and OsPCS2b transcript expressions were observed under cadmium stress. Saccharomyces cerevisiae cells transformed with OsPCS2a exhibited increased cadmium (Cd) and arsenic (As) tolerance and accumulation, unlike the OsPCS2b transformed yeast cells. An intron-containing hairpin RNA-mediated gene silencing was carried out in endosperm-specific manner for efficient down-regulation of OsPCS genes in rice grains. Analysis of the transgenic rice lines grown under metal(loid) stress revealed almost complete absence of both OsPCS1 and OsPCS2 transcripts in the developing seeds coupled with the significant reduction in the content of Cd (~51%) and As (~35%) in grains compared with the non-transgenic plant. Taken together, the findings indicate towards a crucial role played by the tissue-specific alternative splicing and relative abundance of the OsPCS2 gene during heavy metal(loid) stress mitigation in rice plant.

Keywords

Alternative splicing Arsenic stress Cadmium stress Heterologous expression OsPCS2 Phytochelatin synthase RNAi Transgenic rice 

Notes

Acknowledgements

We sincerely thank late Prof. Soumitra K. Sen and Dr. Asitava Basu for their cooperation and help. The authors acknowledge Dr. Agnieszka Golicz for the help during the secondary RNA structure prediction analysis. 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).

Author contributions

Experimental designs and analyses of results were carried out by ND, SB and MKM. ND and SB conducted the experiments and prepared the manuscript. MKM and SB (BCKV) conceived the original research plan. MKM made the necessary corrections in the manuscript and supervised the research work.

Supplementary material

11103_2017_600_MOESM1_ESM.tif (7.2 mb)
Figure S1—Computational prediction of the RNA secondary structure of OsPCS2 transcripts. (A) The graphical representation of the secondary structure of OsPCS2a transcript based on the minimum free energy algorithm and base pairing probabilities. (B) The graphical representation of the secondary structure of OsPCS2b transcript based on the minimum free energy algorithm and base pairing probabilities. (C) The graphical representation of the secondary structure of the retained intron of the OsPCS2b transcript based on the minimum free energy algorithm and base pairing probabilities Supplementary material 1 (TIF 7366 KB)
11103_2017_600_MOESM2_ESM.tif (23.6 mb)
Figure S2—qRT-PCR based relative expression analysis of the OsPCS1 and OsPCS2a transcripts corresponding to the endogenous OsPCS genes in the leaf tissues of transformed rice lines when grown on pot with 10 mg/kg of CdCl2 amended with the soil. The tubulin gene was used as a housekeeping control gene to normalize the expression level. Con-UT: untransformed control rice plant without exogenous Cd stress. Con-Cd: untransformed control rice plant with exogenous Cd stress. ihpL1-Cd, ihpL2-Cd and ihpL3-Cd: the three transgenic rice lines with exogenous Cd stress. The error bars represent the standard deviation of the mean of three independent experiments carried out from three independent mRNA extractions Supplementary material 2 (TIF 24172 KB)

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Department of BiotechnologyIndian Institute of Technology KharagpurKharagpurIndia
  2. 2.Department of GeneticsBidhan Chandra Krishi ViswavidyalayaMohanpurIndia
  3. 3.Plant Molecular Biology and Biotechnology Laboratory, Faculty of Veterinary and Agricultural SciencesUniversity of MelbourneParkvilleAustralia

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