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Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum

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Abstract

Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

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Acknowledgments

Authors thank Dr. P. S. Ahuja, Ex-Director, CSIR-IHBT, Palampur, & Ex-Director-General, CSIR, New Delhi, for the generous gift of fresh P. hexandrum. We are grateful to Prof. S. Roy, Ex-Director, CSIR-IICB for his constant guidance and encouragement. The authors would like to express their gratitude to the Director, CSIR-IICB, Kolkata. This work received financial support from the Council of Scientific and Industrial Research (CSIR), New Delhi. DB, AB and DK acknowledge the CSIR, SH to University Grants Commission, RD to Indian Council of Medical Research for their fellowships. SCb acknowledged the Department of Biotechnology, India for the Ramalingaswami Fellowship and CSIR network project GENESIS (code: BSC0121) for financial support.

Author contributions

Dipto Bhattacharyya and Saptarshi Hazra carried out the experimental work, analyzed the data and framed out the manuscript. Anindyajit Banerjee and Saikat Chakrabarti carried out the docking and related studies. Riddhi Datta developed the transgenic lines. Deepak Kumar helped the seniors to analyze the data. Saikat Chakrabarti helps to draft the manuscript. Sharmila Chattopadhyay designed the experiments and prepared the final manuscript.

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  1. Dipto Bhattacharyya

    Present address: Division of Biotechnology, Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do, 570–752, Republic of Korea

Authors and Affiliations

  1. Plant Biology Lab., Organic and Medicinal Chemistry Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India

    Saptarshi Hazra, Riddhi Datta, Deepak Kumar & Sharmila Chattopadhyay

  2. Structural Biology and Bioinformatics Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India

    Anindyajit Banerjee & Saikat Chakrabarti

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Correspondence to Sharmila Chattopadhyay.

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Dipto Bhattacharyya and Saptarshi Hazra contributed equally to this work.

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Supplementary figures (PDF 5393 KB)

Supplemental Table S1 List of primers used for RT-qPCR analysis. (PDF 18 KB)

Supplemental Table S2 Annotation by BLASTX of Newbler default parameter assembly. (XLS 1464 KB)

Supplemental Table S3 Annotation by BLASTX of Newbler optimized parameter assembly. (XLS 6410 KB)

Supplemental Table S4 The GO annotation of transcripts from Newbler default assembly. (XLS 294 KB)

Supplemental Table S5 The GO annotation of transcripts from Newbler optimized assembly. (XLS 815 KB)

Supplemental Table S6 FPKM values of transcripts from Newbler default parameter assembly. (XLS 469 KB)

Supplemental Table S7 FPKM values of transcripts from Newbler optimized parameter assembly. (XLS 3044 KB)

Supplemental Table S8 Differential expression profiling from Newbler default parameters. (XLS 187 KB)

Supplemental Table S9 Differential expression profiling from Newbler Optimized parameters. (XLS 334 KB)

Supplemental Table S10 KEGG biochemical mapping of transcripts from Newbler default parameter assembly. (XLS 25 KB)

Supplemental Table S11 KEGG biochemical mapping of transcripts from Newbler optimized parameter assembly. (XLS 24 KB)

11103_2016_492_MOESM13_ESM.xls

Supplemental Table S12 List of important domains that may be required for ptox biosynthesis from Newbler default parameter assembly. (XLS 24 KB)

11103_2016_492_MOESM14_ESM.xls

Supplemental Table S13 List of important domains that may be required for ptox biosynthesis from Newbler optimized parameter assembly. (XLS 42 KB)

11103_2016_492_MOESM15_ESM.pdf

Supplemental Table S14 List of important genes that are related phenylpropanoid pathway, obtained from control and MeJA treated cell culture. (PDF 33 KB)

Supplemental Table S15 BLAST result of known protein sequences for secoisolariciresinol dehydrogenase. (PDF 11 KB)

11103_2016_492_MOESM17_ESM.xls

Supplemental Table S16 Complete list of transcription factors identified from transcripts generated by Newbler optimized parameter assembly. (XLS 92 KB)

11103_2016_492_MOESM18_ESM.pdf

Supplemental Table S17 List of sequences and its respective representative’s name used in the phylogenetic tree (PDF 83 KB)

11103_2016_492_MOESM19_ESM.pdf

Supplemental Table S18 Ptox and lignin content in control cell culture and callus of P. hexandrum. Values represent means of three independent replicates ± standard deviations (PDF 68 KB)

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Bhattacharyya, D., Hazra, S., Banerjee, A. et al. Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum . Plant Mol Biol 92, 1–23 (2016). https://doi.org/10.1007/s11103-016-0492-5

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