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Expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in Escherichia coli and Arabidopsis enhances heavy metal(loid)s accumulation

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Abstract

Phytochelatin synthase (PCS) gene encoding key enzyme for heavy metal detoxification and accumulation has been characterised from different sources and used to develop a technology for bioremediation. Past efforts provided limited success and contradictory results. Therefore, functional characterisation of PCS gene from new sources into different target systems is considered as an important task in the area of bioremediation. Earlier, we isolated and functionally characterised PCS gene from an aquatic macrophyte Ceratophyllum demersum L., a metal accumulator aquatic plant. Expression of this gene, CdPCS1, in tobacco enhanced PC synthesis and metal accumulation of transgenic tobacco plants. In the present study, we have expressed CdPCS1 in more diverse systems, Escherichia coli and Arabidopsis, and studied growth and metal accumulation of transgenic organisms. The expression of CdPCS1 in E. coli offered tolerance against cadmium as well as higher accumulation accompanied with PCS1 activity. The expression of CdPCS1 in Arabidopsis showed a significant enhanced accumulation of heavy metal(loid)s in aerial parts without significant difference in growth parameters in comparison to wild-type Arabidopsis plants. Our study suggests that CdPCS1 can be utilised for enhancing bioremediation potential of different organisms using biotechnological approaches.

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Abbreviations

As(III):

Arsenite

As(V):

Arsenate

Cd:

Cadmium

FW:

Fresh weight

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

PC:

Phytochelatin

PCS:

Phytochelatin synthase

WT:

Wild type

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Acknowledgments

Authors acknowledge the Council of Scientific and Industrial Research (CSIR), Government of India, for providing a financial support to carry out a study under Network Project (BSC0107). DS and MT acknowledge the Council of Scientific and Industrial Research and the Indian Council of Medical Research, Government of India, for Senior Research Fellowships, respectively.

Conflict of Interest

All the authors declare that we have no conflict of interest.

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Author notes

  1. Devesh Shukla

    Present address: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

  2. Ravi Kesari

    Present address: Bihar Agriculture University, Bhagalpur, 813210, India

Authors and Affiliations

  1. National Botanical Research Institute (NBRI), Council of Scientific and Industrial Research (CSIR), Rana Pratap Marg, Lucknow, 226001, India

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

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  1. Devesh Shukla
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Correspondence to Prabodh Kumar Trivedi.

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Supplementary Fig. S1

Colony PCR confirmation of construct pGEX4T2:CdPCS1 with gene-specific primers in E. coli BL21 (DE3). Confirmation of recombinant E. coli strain BL21 through colony PCR using CdPCS1 forward and CdPCS1 reverse primers. Numbers 1, 2, 3… represent independent colonies of E. coli. M, ʎ DNA digested HindIII and EcoRI used as a marker (PPT 139 kb)

Supplementary Fig. S2

Comparative display of control or recombinant E. coli growth in the presence or absence of Cd stress. This figure represents the optical density of secondary culture after 5-h growth of control or recombinant E. coli with or without Cd. Growth of E. coli cells (BL21 mock; circle), E. coli cells containing empty vector pGEX-4T2 (triangle) and recombinant plasmid pGEX-4T2-CdPCS1 (rectangle), respectively. Empty and filled markers represent with and without Cd treatment. For detail, please see “Materials and methods”) (PPT 3071 kb)

Supplementary Fig. S3

Fluorescence HPLC chromatograms of the mBBr-labelled E. coli extract to measure PCS activity. Lysate from IPTG-induced cells was used for measuring in vitro enzyme activity in the presence of Cd (100 μM). Quantity of PC2 produced by CdPCS1 enzyme was estimated through fluorescence HPLC (PPT 205 kb)

Supplementary Table S4

List of oligonucleotides used in the study (DOC 32 kb)

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Shukla, D., Kesari, R., Tiwari, M. et al. Expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in Escherichia coli and Arabidopsis enhances heavy metal(loid)s accumulation. Protoplasma 250, 1263–1272 (2013). https://doi.org/10.1007/s00709-013-0508-9

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