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Comparative transcriptome analysis in different tissues of a medicinal herb, Picrorhiza kurroa pinpoints transcription factors regulating picrosides biosynthesis

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Abstract

Transcriptional regulation of picrosides biosynthesis, the iridoid glycosides of an endangered medicinal herb, Picrorhiza kurroa, is completely unknown. P. kurroa plants obtained from natural habitat accumulate higher picrosides than in-vitro cultured plants, which necessitates identification of transcription factors (TFs) regulating their differential biosynthesis. The current study investigates complete spectrum of different TF classes in P. kurroa transcriptomes and discerns their association with picrosides biosynthesis. Transcriptomes of differential picroside-I content shoots and picroside-II content roots were mined for seven classes of TFs implicated in secondary metabolism regulation in plants. Key TFs were identified through in silico transcript abundance and qPCR analysis was performed to confirm transcript levels of TFs under study in differential content tissues and genotypes. Promoter regions of key picrosides biosynthetic pathway genes were explored to hypothesize which TFs can possibly regulate target genes. A total of 131, 137, 107, 82 and 101 transcripts encoding different TFs families were identified in PKS-25, PKS-15, PKSS, PKR-25 and PKSR transcriptomes, respectively. ERF-18, bHLH-104, NAC-25, 32, 94 and SUF-4 showed elevated expression in roots (up to 37 folds) and shoots (up to 195 folds) of plants obtained from natural habitat, indicating their role as activators of picrosides biosynthesis whereas, elevated expression of WRKY-17, 40, 71 and MYB-4 in low picrosides content conditions suggested their down-regulatory role. In silico analysis of key picrosides biosynthetic pathway gene promoter regions revealed binding domains for ERF-18, NAC-25, WRKY-40 and MYB-4. Identification of candidate TFs contributing towards picrosides biosynthesis is a pre-requisite for designing appropriate metabolic engineering strategies aimed at enhancing picrosides content in vitro and in vivo.

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Abbreviations

TF:

Transcription factor

P. kurroa :

Picrorhiza kurroa

PKS-25:

P. kurroa in vitro-cultured shoots grown at 25 °C

PKS-15:

P. kurroa in vitro-cultured shoots grown at 15 °C

PKR-25:

P. kurroa in vitro-cultured roots grown at 25 °C

PKSS:

P. kurroa shoots obtained from natural habitat

PKSR:

P. kurroa roots obtained from natural habitat

P-I and II:

Picroside I and II

HMGR:

Hydroxymethyl glutaryl CoA reductase

ISPD:

2-C-methylerythritol 4-phosphate cytidyl transferase

DXPS:

1-Deoxy-d-xylulose 5-phosphate synthase

PMK:

Phosphomevalonate kinase

HFD:

2 Hydroxyisoflavanone dehydratase

DAHPS:

3-Deoxy-d-arabinoheptulosonate 7-phosphate synthase

ISPE:

4-(Cytidine-5′-diphospho)-2-C-methyl-d-erythritol kinase

CM:

Chorismate mutase

GS:

Geraniol synthase

G10H:

Geraniol 10-hydroxylase

EPSPS:

5-Enolpyruvylshikimic acid-3-phosphate synthase

PAL:

Phenylalanine ammonia lyase

SK:

Shikimate kinase

pkdoubleWRKY:

Double WRKY type transcription factor sequenced from P. kurroa

pkWRKY:

WRKY class transcription factor isolated from P. kurroa

CPRF2:

Common plant regulatory factor 2 (CPRF2) transcription factor

NAP:

NAC like activated by APETALA3/PISTILLATA

COL:

Constans like TF

RSEM:

RNA-Seq by Expectation Maximization

FPKM:

Fragments per kilobase of exon per million fragments mapped

NGS:

Next generation sequencing

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Acknowledgments

The authors are thankful to the Department of Biotechnology, Ministry of Science & Technology, Government of India, for providing research grant in the form of a program support on high value medicinal plants to RSC.

Author contributions

RSC, IV and HS conceived and designed the experiments. IV and TP performed the experiments and analyzed the data. IV was involved in writing the manuscript. RSC and HS revised the manuscript critically and finally approved the manuscript to be published.

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Authors and Affiliations

  1. Department of Biotechnology & Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, 173234, Himachal Pradesh, India

    Ira Vashisht, Tarun Pal, Hemant Sood & Rajinder S. Chauhan

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  1. Ira Vashisht
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  2. Tarun Pal
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  4. Rajinder S. Chauhan
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Correspondence to Rajinder S. Chauhan.

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Vashisht, I., Pal, T., Sood, H. et al. Comparative transcriptome analysis in different tissues of a medicinal herb, Picrorhiza kurroa pinpoints transcription factors regulating picrosides biosynthesis. Mol Biol Rep 43, 1395–1409 (2016). https://doi.org/10.1007/s11033-016-4073-0

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