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


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.


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.


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



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)


  1. Bhattacharya S, Chattopadhyaya B, Koduru L, Das N, Maiti MK (2014) Bran-specific expression of Brassica juncea microsomal ω-3 desaturase gene (BjFad3) improves the nutritionally desirable ω-6:ω-3 fatty acid ratio in rice bran oil. Plant Cell Tissue Organ 119(1):117–129CrossRefGoogle Scholar
  2. Bhattacharya S, Sinha S, Das N, Maiti MK (2015) Increasing the stearate content in seed oil of Brassica juncea by heterologous expression of MlFatB affects lipid content and germination frequency of transgenic seeds. Plant Physiol Biochem 96:345–355CrossRefPubMedGoogle Scholar
  3. Bhattacharya S, Das N, Maiti MK (2016) Cumulative effect of heterologous AtWri1 gene expression and endogenous BjAGPase gene silencing increases seed lipid content in Indian mustard Brassica juncea. Plant Physiol Biochem 107:204–213CrossRefPubMedGoogle Scholar
  4. Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336CrossRefPubMedGoogle Scholar
  5. Boc A, Diallo AB, Makarenkov V (2012) T-REX: a web server for inferring, validating and visualizing phylogenetic trees and networks. Nucleic Acids Res 40:W573–W579CrossRefPubMedPubMedCentralGoogle Scholar
  6. Brunetti P, Zanella L, Proia A, De Paolis A, Falasca G, Altamura MM, Sanita` di Toppi L, Costantino P, Cardarelli M (2011) Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1. J Exp Bot 62:5509–5519CrossRefPubMedPubMedCentralGoogle Scholar
  7. Buratti E, Baralle FE (2004) Influence of RNA secondary structure on the pre-mRNA splicing process. Mol Cell Biol 24:10505–10514CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cazalé AC, Clemens S (2001) Arabidopsis thaliana expresses a second functional phytochelatin synthase. FEBS Lett 507:215–219CrossRefPubMedGoogle Scholar
  9. Cheng F, Zhao N, Xu H, Li Y, Zhang W, Zhu Z, Chen M (2006) Cadmium and lead contamination in japonica rice grains and its variation among the different locations in southeast China. Sci Total Environ 359:156–166CrossRefPubMedGoogle Scholar
  10. Clemens S, Kim EJ, Neumann D, Schroeder JI (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 18:3325–3333CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cobbett CS (2000) Phytochelatins and their role in heavy metal detoxification. Plant Physiol 123:825–833CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cobbett CS, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Physiol Plant Mol Biol 53:159–182CrossRefGoogle Scholar
  13. Das N, Bhattacharya S, Maiti MK (2016) Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation. Plant Physiol Biochem 105:297–309CrossRefPubMedGoogle Scholar
  14. Deshler JO, Rossi JJ (1991) Unexpected point mutations activate cryptic 3′ splice sites by perturbing a natural secondary structure within a yeast intron. Genes Dev 5:1252–1263CrossRefPubMedGoogle Scholar
  15. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  16. Ellis JD, Barrios-Rodiles M, Colak R, Irimia M, Kim T, Calarco JA, Wang X, Pan Q, O’Hanlon D, Kim PM, Wrana JL, Blencowe BJ (2012) Tissue-specific alternative splicing remodels protein-protein interaction networks. Mol Cell 46:884–892CrossRefPubMedGoogle Scholar
  17. Filichkin SA, Mockler TC (2012) Unproductive alternative splicing and nonsense mRNAs: a widespread phenomenon among plant circadian clock genes. Biol Direct 7:20CrossRefPubMedPubMedCentralGoogle Scholar
  18. Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong WK, Mockler TC (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20:45–58CrossRefPubMedPubMedCentralGoogle Scholar
  19. Fits VL, Deakin EA, Hoge JH, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502CrossRefPubMedGoogle Scholar
  20. Gasic K, Korban SS (2007) Expression of Arabidopsis phytochelatin synthase in Indian mustard (Brassica juncea) plants enhances tolerance for Cd and Zn. Planta 225:1277–1285CrossRefPubMedGoogle Scholar
  21. Gisbert C, Ros R, Haro AD, Walker DJ, Bernal MP, Serrano R, Navarro-Aviñó J (2003) A plant genetically modified that accumulates Pb is especially promising for phytoremediation. Biochem Biophys Res Commun 303:440–445CrossRefPubMedGoogle Scholar
  22. Goguel V, Wang Y, Rosbash M (1993) Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing. Mol Cell Biol 13:6841–6848CrossRefPubMedPubMedCentralGoogle Scholar
  23. Graveley BR (2001) Alternative splicing: increasing diversity in the proteomic world. Trends Genet 17:100–107CrossRefPubMedGoogle Scholar
  24. Grill E, Winnacker EL, Zenk MH (1987) Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proc Natl Acad Sci USA 84(2):439–443CrossRefPubMedPubMedCentralGoogle Scholar
  25. Grill E, Löffler S, Winnacker EL, Zenk MH (1989) Phytochelatins, the heavy-metal binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc Natl Acad Sci USA 86:6838–6842CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ha SB, Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, Goldsbrough PB, Cobbett CS (1999) Phytochelatin synthase genes from Arabidopsis and the yeast, Schizosaccharomyces pombe. Plant Cell 11:1153–1164CrossRefPubMedPubMedCentralGoogle Scholar
  27. Herawati N, Suzuki S, Hayashi K, Rivai IF, Koyama H (2000) Cadmium, copper, and zinc levels in rice and soil of Japan, Indonesia, and China by soil type. Bull Environ Contam Toxicol 64:33–39CrossRefPubMedGoogle Scholar
  28. Hori K, Watanabe Y (2007) Context analysis of termination codons in mRNA that are recognized by plant NMD. Plant Cell Physiol 48:1072–1078CrossRefPubMedGoogle Scholar
  29. Howden R, Goldsbrough PB, Andersen CR, Cobbett CS (1995) Cadmium-sensitive cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107:1059–1066CrossRefPubMedPubMedCentralGoogle Scholar
  30. Johanning J, Strasdeit H (1998) A coordination-chemical basis for the biological function of the phytochelatins. Angew Chem Int Ed 37(18):2464–2466CrossRefGoogle Scholar
  31. Kühnlenz T, Schmidt H, Uraguchi S, Clemens S (2014) Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the AtPCS1-deficient cad1-3 mutant on Cd-contaminated soil. J Exp Bot 65(15):4241–4253CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lareau LF, Green RE, Bhatnagar RS, Brenner SE (2004) The evolving roles of alternative splicing. Curr Opin Struct Biol 14:273–282CrossRefPubMedGoogle Scholar
  33. Lee S, Moon JS, Ko T, Petros D, Golsbrough PB, Korban SS (2003) Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol 131:656–663CrossRefPubMedPubMedCentralGoogle Scholar
  34. Li Y, Dhankher OM, Carreira L, Lee D, Chen A, Schroeder JI, Balish RS, Meagher RB (2004) Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity. Plant Cell Physiol 45:1787–1797CrossRefPubMedGoogle Scholar
  35. Li Y, Dankher OP, Carreira L, Smith AP, Meagher RB (2006) The shoot-specific expression of gamma-glutamylcysteine synthetase directs the long-distance transport of thiol-peptides to roots conferring tolerance to mercury and arsenic. Plant Physiol 141:288–298CrossRefPubMedPubMedCentralGoogle Scholar
  36. Li JC, Guo JB, Xu WZ, Ma M (2007) RNA interference-mediated silencing of phytochelatin synthase gene reduce cadmium accumulation in rice seeds. J Integr Plant Biol 49:1032–1037CrossRefGoogle Scholar
  37. Li AM, Yu BY, Chen FH, Gan HY, Yuan JG, Qiu R, Huang JC, Yang ZY, Xu ZF (2009) Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms. Int J Mol Sci 10:3269–3282CrossRefPubMedPubMedCentralGoogle Scholar
  38. Lombi E, Scheckel KG, Pallon J, Carey AM, Zhu YG, Meharg AA (2009) Speciation and distribution of As and localization of nutrients in rice grains. New Phytol 184:193–201CrossRefPubMedGoogle Scholar
  39. Lorenz R, Bernhart SH, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL (2011) ViennaRNA Package 2.0. Algorithms. Mol Biol 6:26Google Scholar
  40. Marrs KA, Walbot V (1997) Expression and RNA splicing of the maize glutathione S-transferase Bronze2 gene is regulated by cadmium and other stresses. Plant Physiol 113:93–102CrossRefPubMedPubMedCentralGoogle Scholar
  41. Martínez M, Bernal P, Almela C, Vélez D, García-Augustín P, Serrano R, Navarro-Aviñó J (2006) An engineered plant that accumulates higher levels of heavy metals than Thlaspi caerulescens, with yields of 100 times more biomass in mine soils. Chemosphere 64:478–485CrossRefPubMedGoogle Scholar
  42. Mayeda A, Munroe SH, Cáceres JF, Krainer AR (1994) Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J 15:5483–5495Google Scholar
  43. Meharg AA, Williams PN, Adomako E, Lawgali YY, Deacon C, Villada A, Cambell RCJ, Sun GX, Zhu YG, Feldmann J, Raab A, Zhao FJ, Islam MR, Hossain S, Yanai J (2009) Geographical variation in total and inorganic arsenic content of polished (white) rice. Environ Sci Technol 43:1612–1617CrossRefPubMedGoogle Scholar
  44. Pomponi M, Censi V, Di Girolamo V, De Paolis A, di Toppi LS, Aromolo R, Costantino P, Cardarelli M (2006) Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2 + tolerance and accumulation but not translocation to the shoot. Planta 223:180–190CrossRefPubMedGoogle Scholar
  45. Qu LQ, Xing YP, Liu WX, Xu XP, Song YR (2008) Expression pattern and activity of six glutelin gene promoters in transgenic rice. J Exp Bot 59:2417–2424CrossRefPubMedCentralGoogle Scholar
  46. Ramos J, Clemente MR, Naya L, Loscos J, Pérez-Rontomé C, Sato S, Tabata S, Becana M (2007) Phytochelatin synthases of the model legume Lotus japonicus. A small multigene family with differential response to cadmium and alternatively spliced variants. Plant Physiol 143:1110–1118CrossRefPubMedPubMedCentralGoogle Scholar
  47. Ramos J, Naya L, Gay M, Abian J, Becana M (2008) Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus. Plant Physiol 148:536–545CrossRefPubMedPubMedCentralGoogle Scholar
  48. Rauser WE (1995) Phytochelatins and related peptides: structure, biosynthesis, and function. Plant Physiol 109:1141–1149CrossRefPubMedPubMedCentralGoogle Scholar
  49. Ray D, Williams DL (2011) Characterization of the phytochelatin synthase of Schistosoma mansoni. PLoS Negl Trop Dis 5:e1168CrossRefPubMedPubMedCentralGoogle Scholar
  50. Reddy AS (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58:267–294CrossRefPubMedGoogle Scholar
  51. Rogan N, Serafimovski T, Dolenec M, Tasev G, Dolenec T (2009) Heavy metal contamination of paddy soils and rice (Oryza sativa L.) from Kočani Field (Macedonia). Environ Geochem Health 31:439–451CrossRefPubMedGoogle Scholar
  52. Romanyuk ND, Rigden DJ, Vatamaniuk OK, Lang A, Cahoon RE, Jez JM, Rea PA (2006) Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase. Plant Physiol 141(3):858–869CrossRefPubMedPubMedCentralGoogle Scholar
  53. Ruotolo R, Peracchi A, Bolchi A, Infusini G, Amoresano A, Ottonello S (2004) Domain organization of phytochelatin synthase. Functional properties of truncated enzyme species identified by limited proteolysis. J Biol Chem 279:14686–14693CrossRefPubMedGoogle Scholar
  54. Sauge-Merle S, Cuine S, Carrier P, Lecomte-Pradines C, Luu DT, Peltier G (2003) Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase. Appl Environ Microbio 69:490–494CrossRefGoogle Scholar
  55. Schmöger MEV, Oven M, Grill E (2000) Detoxification of arsenic by phytochelatins in plants. Plant Physiol 122:793–802CrossRefPubMedPubMedCentralGoogle Scholar
  56. Shri M, Dave R, Dwivedi S, Shukla D, Kesari R, Tripathi RD, Trivedi PK, Chakrabarty D (2014) Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain. Sci Rep 4:5784CrossRefPubMedPubMedCentralGoogle Scholar
  57. Song WY, Yamaki T, Yamaji N, Ko D, Jung KH, Fujii-Kashino M, An G, Martinoia E, Lee Y, Ma JF (2014) A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain. Proc Natl Acad Sci USA 111:15699–15704CrossRefPubMedPubMedCentralGoogle Scholar
  58. Syed NH, Kalyna M, Marquez Y, Barta A, Brown JW (2012) Alternative splicing in plants–coming of age. Trends Plant Sci 17:616–623CrossRefPubMedPubMedCentralGoogle Scholar
  59. Trotta E (2014) On the normalization of the minimum free energy of RNAs by sequence length. PLoS ONE 9:e113380CrossRefPubMedPubMedCentralGoogle Scholar
  60. Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis : isolation and in vitroreconstitution. Proc Natl Acad Sci USA 96:7110–7115CrossRefPubMedPubMedCentralGoogle Scholar
  61. Vatamaniuk OK, Mari S, Lang A, Chalasani S, Demkiv LO, Rea PA (2004) Phytochelatin synthase, a dipeptidyltransferase that undergoes multisite acylation with γ-glutamylcysteine during catalysis. J Biol Chem 279(21):22449–22460CrossRefPubMedGoogle Scholar
  62. Vivares D, Arnoux P, Pignol D (2005) A papain-like enzyme at work: native and acyl-enzyme intermediate structures in phytochelatin synthesis. Proc Natl Acad Sci USA 102:18848–18853CrossRefPubMedPubMedCentralGoogle Scholar
  63. Vogeli-Lange R, Wagner GJ (1990) Subcellular localization of cadmium-binding peptides in tobacco leaves. Implications of a transport function for cadmium-binding peptides. Plant Physiol 92:1086–1093CrossRefPubMedPubMedCentralGoogle Scholar
  64. Wang BB, Brendel V (2006) Genome-wide comparative analysis of alternative splicing in plants. Proc Natl Acad Sci USA 103:7175–7180CrossRefPubMedPubMedCentralGoogle Scholar
  65. Williams PN, Lei M, Sun GX, Huang Q, Lu Y, Deacon C, Meharg AA, Zhu YG (2009) Occurrence and partitioning of cadmium, arsenic and lead in mine impacted paddy rice: Hunan, China. Sci Total Environ 43:637–642CrossRefGoogle Scholar
  66. Wu J, Kang JH, Hettenhausen C, Baldwin IT (2007) Nonsense mediated mRNA decay (NMD) silences the accumulation of aberrant trypsin proteinase inhibitor mRNA in Nicotiana attenuata. Plant J 51:693–706CrossRefPubMedGoogle Scholar
  67. Zenk MH (1996) Heavy metal detoxification in higher plants - a review. Gene 179:21–30CrossRefPubMedGoogle Scholar
  68. Zhang BH, Pan XP, Cox SB, Cobb GP, Anderson TA (2006) Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci 63:246–254CrossRefPubMedGoogle Scholar
  69. Zheng R, Chen Z, Cai C, Tie B, Liu X, Reid BJ, Huang Q, Lei M, Sun G, Baltrėnaitė E (2015) Mitigating heavy metal accumulation into rice (Oryza sativa L.) using biochar amendment-a field experiment in Hunan, China. Environ Sci Pollut Res 22:11097–11108CrossRefGoogle Scholar
  70. Zhu J, Krainer AR (2000) Pre-mRNA splicing in the absence of an SR protein RS domain. Genes Dev 14:3166–3178CrossRefPubMedPubMedCentralGoogle Scholar
  71. Zhu YG, Williams PN, Meharg AA (2008) Exposure to inorganic arsenic from rice: a global health issue? Environ Pollut 154:169–171CrossRefPubMedGoogle Scholar

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

Personalised recommendations