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Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco

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Abstract

Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757 bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C. demersum.

Key message

Heterologous expression of PCS gene from C. demersum complements Arabidopsis cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.

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Abbreviations

As(V):

Arsenate

Cd:

Cadmium

CdPCS1/cad1-3 :

cad1-3 mutant plants transformed with CdPCS1 construct

γ-EC:

Gamma-glutamylcysteine

FW:

Fresh weight

GSH:

Glutathione

PC:

Phytochelatin

PCS:

Phytochelatin synthase

WT:

Wild type

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Acknowledgments

We thank Prof. Christopher Cobbett (University of Melbourne, Australia) for providing cad1-3 seeds. DS, RK and SM acknowledge Council of Scientific and Industrial Research, Govt. of India for Senior Research Fellowships. The authors are grateful to the Department of Biotechnology, New Delhi for providing financial support to carry out the work.

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Authors and Affiliations

  1. CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226 001, India

    Devesh Shukla, Ravi Kesari, Seema Mishra, Sanjay Dwivedi, Rudra Deo Tripathi, Pravendra Nath & Prabodh Kumar Trivedi

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  1. Devesh Shukla
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  2. Ravi Kesari
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  3. Seema Mishra
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  4. Sanjay Dwivedi
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  7. Prabodh Kumar Trivedi
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Correspondence to Prabodh Kumar Trivedi.

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Communicated by P. Kumar.

Electronic supplementary material

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Supplementary material 1 (DOC 37 kb)

299_2012_1283_MOESM2_ESM.pptx

Fig. S1 Schematic representation of sequence alignment of PCS protein sequences from selected organisms such as Arabidopsis thaliana (At) and Triticum aestivum (Ta) chosen for plants; Schizosaccharomyces pombe (Sp) chosen for fungi and C. demersum (Cd) chosen as the case of present study. Black vertical bars denote cysteine residues, black vertical bars with asterisks denote conserved cysteine residues present in N-terminal. Red vertical bar with asterisks denotes conserved His-162 and blue vertical bar denotes conserved Asp-180, considered as an essential part of catalytic domain of the enzyme. Supplementary material 2 (PPT 227 kb)

299_2012_1283_MOESM3_ESM.pptx

Fig. S2 Fluorescence HPLC chromatograms of the mBBr-labeled plant extracts from WT and transgenic lines. Root extracts of WT and transgenic lines grown in absence of heavy metals (0 μM) were derivatized with mBBr and separated by HPLC. Peaks corresponding to cysteine, γ-EC, GSH, PC2, PC3 and PC4 standards are indicated in the chromatogram. Other unknown non-protein thiols (Peaks A-E) which are differentially accumulated in transgenic lines with respect to WT are marked. Supplementary material 3 (PPT 187 kb)

299_2012_1283_MOESM4_ESM.pptx

Fig. S3 Fluorescence HPLC chromatograms of the mBBr-labeled plant extracts from WT and transgenic lines. Root extracts of WT and transgenic lines exposed on Cd (200 μM) for 3 days were derivatized with mBBr and separated by HPLC. Peaks corresponding to cysteine, γ-EC, GSH, PC2, PC3 and PC4 standards are indicated in the chromatogram. Other unknown non-protein thiols (Peaks A-E) which are differentially accumulated in transgenic lines with respect to WT are marked. Supplementary material 4 (PPT 328 kb)

299_2012_1283_MOESM5_ESM.pptx

Fig. S4 Fluorescence HPLC chromatograms of the mBBr-labeled plant extracts from WT and transgenic lines. Root extracts of WT and transgenic lines exposed on AsV (200 μM) for 3 days were derivatized with mBBr and separated by HPLC. Peaks corresponding to cysteine, γ-EC, GSH, PC2, PC3 and PC4 standards are indicated in the chromatogram. Other unknown non-protein thiols (Peaks A-E) which are differentially accumulated in transgenic lines with respect to WT are marked. Supplementary material 5 (PPT 180 kb)

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Shukla, D., Kesari, R., Mishra, S. et al. Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco. Plant Cell Rep 31, 1687–1699 (2012). https://doi.org/10.1007/s00299-012-1283-3

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