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Molecular characterization of DWF1 from Withania somnifera (L.) Dunal: its implications in withanolide biosynthesis

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Abstract

Present study describes isolation and molecular characterization of DWF1 gene from Withania somnifera and also elucidates its role in withanolide biosynthesis. Using the degenerate-reverse transcriptase and RACE strategy full length cDNA of DWF1 was isolated from W. somnifera and subsequently cloned in pGEX-4T-2 vector and expressed in E. coli. Effect of various abiotic and biotic chemicals such as methyl jasmonate (MeJA), salicylic acid (SA), 2,4-dichlorophenoxyacetic acid (2,4-D) and microbe-derived exogenous elicitor yeast extract (YE) was analyzed to establish any correlation between expression levels of transcript and withanolide accumulation. Effects on the transcript levels of WsDWF1 with each progressing ontogenic stages of W. somnifera was also carried out. Tissue-specific expression studies analyzing expression of WsDWF1 in various tissues such as leaf, root and stalk was also carried out. Full length cDNA of WsDWF1 contained an ORF of 1707 bp and encoded 65.8 kDa protein. Based upon elicitation studies using various abiotic and biotic chemicals such MeJA, SA, 2,4-D and YE, a positive correlation between withanolide accumulation and transcript profile of WsDWF1 was observed. Tissue-specific expression studies suggested higher expression of WsDWF1 in leaves followed by stalk and root tissues. A uniform increase in the transcript levels of WsDWF1 with each progressing developemental stage indicated increased substrate pool for phytosterol biosynthesis. Use of YE a known inhibitor of oxidosqualene cyclases (OSCs), showed correspondence with the increased transcript levels of WsDWF1 and an enhanced withanolide accumulation, possibly by re-routing of metabolite flux towards the downstream phytosterol biosynthesis. Present work describes WsDWF1 as a new player in withanogenesis, which can open up a fresh prospect for pathway engineering of W. somnifera.

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Abbreviations

WS-3:

Withaferin A

DWF1:

DIMUNUTO/DWARF1

WS-1:

Withanolide A

SA:

Salicylic acid

MeJA:

Methyl jasmonate

2,4-D:

2,4-dichlorophenoxyacetic acid

YE:

Yeast extract

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Acknowledgments

This work was supported by a grant from the Council of Scientific and Industrial Research (CSIR), New Delhi under Network Project BSC-0108. S. Razdan, N. Dhar, S. Rana, W. W. Bhat are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for Senior Research Fellowship (CSIR-SRF). S. A. Pandith is grateful to University Grants Commission, New Delhi for Senior Research Fellowship (UGC-SRF). This manuscript represents institutional communication number IIIM/1817/2015.

Author contributions

Conceived and designed the experiments: SKL, RV. Performed the experiments: S. Razdan, WWB, ND S. Rana, SAP, TAW. Analyzed the data: S. Razdan, S. Rana, SKL. Contributed reagents/materials/analysis tools: RV and SKL. Wrote the paper: S. Razdan and SKL.

Author information

Author notes

  1. Wajid Waheed Bhat

    Present address: Biotransformation, Scion Research NZ- Crown Research Institute, 49 Sala Street, Rotorua, 3046, New Zealand

  2. Niha Dhar

    Present address: Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore

  3. Satiander Rana

    Present address: Genetics, Development & Cell Biology, NSF Engineering Research Center for Bio-renewable Chemicals, Biorenewables Research Laboratory, Iowa State University, Ames, IA, 50011, USA

Authors and Affiliations

  1. Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India

    Sumeer Razdan, Wajid Waheed Bhat, Niha Dhar, Satiander Rana, Shahzad A. Pandith, Tareq A. Wani & Surrinder K. Lattoo

  2. Medicinal Chemistry, CSIR–Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India

    Ram Vishwakarma

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Correspondence to Surrinder K. Lattoo.

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Fig. S1

(A) Time courses of WsDWF1 expression in micro propagated Withania somnifera. The elicitation was done by yeast extract (YE; 0.1 % w/v). Experiments were performed in triplicate with similar results; error bars indicate ± standard deviation of the mean. (B) 0.1 %w/v yeast extract (YE) at different time courses. Variation in two key withanolides - withanolide A (WS-1) and withaferin A (WS-3) was confirmed by HPLC analysis at 6, 12, 24 and 48 h. All values obtained were means of triplicate with standard errors. Time-course accumulation of WS-1 and WS-3 was statistically significant at *p < 0.01level and **p < 0.01 levels (JPEG 183 kb)

Fig. S2

(A) Tissue-specific real-time expression analysis. Quantitative estimation of the expression of WsDWF1 in leaf, roots and stalk of Withania somnifera. Values are means, with standard errors indicated by bars, representing three independent biological samples, each with three technical replicates. Differences were scored as statistical significance at *p < 0.05 and **p < 0.01 levels. S2. (B) Transcript profiles of WsDWF1 during various ontogenetic stages of Withania somnifera. a) vegetative, b) flowering c) fruit set d) fruit maturation e) over maturation stages (JPEG 89 kb)

Fig. S3

Multiple alignment of the deduced amino acid sequence of WsDWF1 with the known DWF1 sequences of other plant families by Clustal W multiple alignment tool. The aligned sequences are from Solanum tuberosum (CBP07442), Solanum lycopersicum(NP_001234550), Ricinus communis (XP_002520604), Populus triocarpa (XP_0023315056), Arabidopsis thaliana (NP_188616), Zea mays (AFW69640), Theobroma cacao (XP_007045589), Oryza sativa (AAM01136), Aegilops tauschii (EMT16029), Nicotiana tabacum (AIL30429), Brassica napus (CDY42207), Zinnia Violacea (BAE16980) (JPEG 731 kb)

Fig. S4

Prediction of three dimensional structure of WsDWF1 using Phyre2 bioinformatics tool. A: Predicted structure of WsDWF1 was based upon 6 protein templates (C4ml8C, C4fdoA, C4bc9C, C3bw7A, C3rjaA, C2bvfA). B: Prediction of ligand binding sites using 3D ligand site web server. ADP, ZEA and FAD heterogens, ligand binding interactions with the predicted binding sites are shown in the picture (JPEG 340 kb)

Fig. S5

Neighbour joining phylogenetic tree constructed from the deduced amino acid sequences of various organisms using MEGA 5.05. Numbers above the branches indicate bootstrap values. Members of the Solanaceae family including W. somnifera are present in a separate clade (JPEG 275 kb)

Fig. S6

Hydropathy prediction of the WsDWF1 protein usingTMpred program. Presence of two transmembrane helices from 26 aa to 44 aa and 208 aa to 232 aa is depicted in the figure (JPEG 132 kb)

Fig. S7

HPLC chromatograms of withanolide standards (WS-1; withanolide A and WS-3; withaferin-A) (A), extract of control samples (B), methyl jasmonate (MeJA; C and D), salicylic acid (SA; E and F) (JPEG 164 kb)

Fig. S8

HPLC analysis of standard withanolide A (WS-1) and withaferin A (WS-3). Quantitation of withanolides at (A) 6 h and (B) 48 h from 0.1 % w/v yeast extract (YE) treated micro-shoots of Withania somnifera by HPLC (JPEG 144 kb)

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Razdan, S., Bhat, W.W., Dhar, N. et al. Molecular characterization of DWF1 from Withania somnifera (L.) Dunal: its implications in withanolide biosynthesis. J. Plant Biochem. Biotechnol. 26, 52–63 (2017). https://doi.org/10.1007/s13562-016-0359-5

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