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Metabolic shift from withasteroid formation to phenylpropanoid accumulation in cryptogein-cotransformed hairy roots of Withania somnifera (L.) Dunal

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Abstract

Cotransformed hairy roots containing a gene that encodes a fungal elicitor protein, β-cryptogein, were established in Withania somnifera, a medicinal plant widely used in Indian systems of medicine. To find out whether β-cryptogein protein endogenously elicits the pathway of withasteroid biosynthesis, withaferin A and withanolide A contents along with transcript accumulation of farnesyl pyrophosphate (FPP) synthase, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), and sterol glycosyltransferase (SGT) were analyzed in both cryptogein-cotransformed and normal hairy roots of W. somnifera. It was observed that the withaferin A and withanolide A contents were drastically higher in normal hairy roots than cryptogein-cotransformed ones. Similar trends were also observed on the levels of transcript accumulation. Subsequently, the enzyme activity of phenylalanine ammonia lyase (PAL), one of the key enzymes of phenylpropanoid pathway, was measured in both cryptogein-cotransformed and normal hairy roots of W. somnifera along with the levels of PAL transcript accumulation. Upliftment of PAL activity was observed in cryptogein-cotransformed hairy roots as compared to the normal ones, and the PAL expression also reflected a similar trend, i.e., enhanced expression in the cryptogein-cotransformed lines. Upliftment of wall-bound ferulic acid accumulation was also observed in the cryptogein-cotransformed lines, as compared to normal hairy root lines. Thus, the outcome of the above studies suggests a metabolic shift from withanolide accumulation to phenylpropanoid biosynthesis in cryptogein-cotransformed hairy roots of W. somnifera.

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Acknowledgments

S Jha thanks David Tepfer of INRA, Versailles (France) for providing the Agrobacterium rhizogenes 9402 strain harboring a binary vector (pBIN19) containing the β-cryptogein gene. B Sil thanks the Council of Scientific and Industrial Research (CSIR), India, for the award of an individual senior research fellowship (SRF-NET). Facilities created from a research grant (SERB/SR/S0/PS/18/2011 to A Mitra and S Jha) of the Science and Engineering Research Board, Department of Science and Technology, India, for a related project, were utilized in this work.

Conflict of interest

The authors declare that they have no conflict of interest.

Author contribution

AM, BS, and CM conceived and designed research. BS did all the experiments. BS and CM analyzed the data. SJ guided the genetic transformation experiments and took the overall responsibility. AM wrote the final manuscript. All authors read and approved the manuscript.

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Authors and Affiliations

  1. Centre for Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700 019, India

    Bipradut Sil & Sumita Jha

  2. Natural Product Biotechnology Group, Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur, 721 302, India

    Chiranjit Mukherjee & Adinpunya Mitra

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  1. Bipradut Sil
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  2. Chiranjit Mukherjee
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  3. Sumita Jha
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  4. Adinpunya Mitra
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Corresponding author

Correspondence to Adinpunya Mitra.

Additional information

Handling Editor: Peter Nick

BS and CM contributed equally to this work.

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Supplementary Fig. 1

PCR amplification of cryptogein gene in normal hairy roots A) and crypt-cotransformed B) hairy roots of W. somnifera. (C) Positive control, A. rhizogenes strain LBA9402 harboring pBIN19-CRYPT binary vector; (M) Molecular marker (Gene Ruler 100 bp DNA ladder). (GIF 56 kb)

High Resolution Image (TIFF 485 kb)

Supplementary Fig. 2

Amino acid sequence alignment of putative PAL gene of W. somnifera and corresponding sequences from other plant. Gene Bank accession numbers are mentioned beside each genus. (GIF 371 kb)

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Sil, B., Mukherjee, C., Jha, S. et al. Metabolic shift from withasteroid formation to phenylpropanoid accumulation in cryptogein-cotransformed hairy roots of Withania somnifera (L.) Dunal. Protoplasma 252, 1097–1110 (2015). https://doi.org/10.1007/s00709-014-0743-8

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