Skip to main content
SpringerLink
Log in

Complexity of gene paralogues resolved in biosynthetic pathway of hepatoprotective iridoid glycosides in a medicinal herb, Picrorhiza kurroa through differential NGS transcriptomes

  • Original Article
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Picrorhiza kurroa is a medicinal herb with diverse pharmacological applications due to the presence of iridoid glycosides, picroside-I (P-I), and picroside-II (P-II), among others. Any genetic improvement in this medicinal herb can only be undertaken if the biosynthetic pathway genes are correctly identified. Our previous studies have deciphered biosynthetic pathways for P-I and P-II, however, the occurrence of multiple copies of genes has been a stumbling block in their usage. Therefore, a methodological strategy was designed to identify and prioritize paralogues of pathway genes associated with contents of P-I and P-II. We used differential transcriptomes varying for P-I and P-II contents in different tissues of P. kurroa. All transcripts for a particular pathway gene were identified, clustered based on multiple sequence alignment to notify as a representative of the same gene (≥ 99% sequence identity) or a paralogue of the same gene. Further, individual paralogues were tested for their expression level via qRT-PCR in tissue-specific manner. In total 44 paralogues in 14 key genes have been identified out of which 19 gene paralogues showed the highest expression pattern via qRT-PCR. Overall analysis shortlisted 6 gene paralogues, PKHMGR3, PKPAL2, PKDXPS1, PK4CL2, PKG10H2 and PKIS2 that might be playing role in the biosynthesis of P-I and P-II, however, their functional analysis need to be further validated either through gene silencing or over-expression. The usefulness of this approach can be expanded to other non-model plant species for which transcriptome resources have been generated.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig.4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

4CL:

4- coumarate Ligase

CAM:

Caffeic acid O-methyltransferase

CM:

Chorismate mutase

C4H:

Cinnamate 4-hydroxylase

DAHPS:

3-deoxy-d-arabino heptulosonate 7-phosphate synthase

DXPS:

1-Deoxy-d-xylulose-5-phosphate synthase

EPSPS:

5-Enol-pyruvylshikimate-phosphate synthase

G10H:

Geraniol 10-hydroxylase

HMGR:

3-Hydroxy-3-methylglutaryl-coenzyme a reductase

IS:

Iridoid synthase

ISPD:

2C-methyl-d-erythritol 4-phosphate cytidylyltransferase

MECPS:

2C-methyl-d-erythritol-cyclodiphosphate synthase

PAL:

Phenylalanine ammonia-lyase

PMK:

Phosphomevalonate kinase

PK:

Picrorhiza kurroa

References

  • Cass CL, Peraldi A, Dowd PF, Mottiar Y, Santoro N, Karlen SD, Bukhman YV (2015) Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium. J Exp Bot 66:4317–4335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dai Z, Guanghong C, Shu-Feng Z, Xianan Z, Luqi H (2011) Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation. J Plant Physiol 168:148–157

    Article  CAS  PubMed  Google Scholar 

  • Eberhard J, Raesecke HR, Schmid J, Amrhein N (1993) Cloning and expression in yeast of a higher plant Chorismate Mutase. Molecular cloning, sequencing of the CDNA and characterization of the Arabidopsis thaliana enzyme expressed in yeast. FEBS Lett 334:233–236

    Article  CAS  PubMed  Google Scholar 

  • Gao S, Juan L, Xuefen L, Zhongxiang D, Yingjun L, Xiaofen S, Kexuan T (2006) Molecular cloning, characterization and functional analysis of a 2C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase gene from Ginkgo biloba. J Biochem Mol Biol 39:502–510

    CAS  PubMed  Google Scholar 

  • Goers SK, Jensen RA (1984) The differential allosteric regulation of two chorismate-mutase Isoenzymes of Nicotiana silvestris. Planta 162:117–124

    Article  CAS  PubMed  Google Scholar 

  • Görlach J, Beck A, Henstrand JM, Handa AK, Herrmann KM, Schmid J, Amrhein N (1993) Differential expression of tomato (Lycopersicon esculentum) genes encoding shikimate pathway isoenzymes. I. 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase. Plant Mol Biol 23:697–706

    Article  PubMed  Google Scholar 

  • Gui J, Junhui S, Li L (2011) Functional characterization of evolutionarily divergent 4-coumarate: Coenzyme A ligases in rice. Plant Physiol 157:574–586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harker M, Holmberg N, Clayton JC, Gibbard CL, Wallace AD, Rawlins S, Hellyer SA, Lanot A, Safford R (2003) Enhancement of seed phytosterol levels by expression of an N-terminal truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase. Plant Biotechnol J 1:113–121

    Article  CAS  PubMed  Google Scholar 

  • Hsieh M-H, Chiung-Yun C, Shih-Jui H, Ju-Jiun C (2008) Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in IspD and IspE albino mutants in Arabidopsis. Plant Mol Biol 66:663–673

    Article  CAS  PubMed  Google Scholar 

  • Iida T, Takehiko K (2019) How do cells count multi-copy genes? ‘Musical Chair’ model for preserving the number of RDNA copies. Curr Genet 65:883–885

    Article  CAS  PubMed  Google Scholar 

  • Kant K, Walia M, Agnihotri VK, Pathania V, Singh B (2013) Evaluation of antioxidant activity of Picrorhiza kurroa (leaves) extracts. Indian J Pharm Sci 75:324–329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kevei Z, Lougnon G, Mergaert P, Gábor V, Horváth KA, Jayaraman DK, Zaman N (2007) 3-Hydroxy-3-methylglutaryl coenzyme A reductase1 interacts with NORK and is crucial for nodulation in Medicago truncatula. Plant Cell 19:3974–3989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar V, Sood H, Sharma M, Chauhan RS (2013) A proposed biosynthetic pathway of picrosides linked through the detection of biochemical intermediates in the endangered medicinal herb Picrorhiza kurroa. Phytochem Anal 24:598–602

  • Kumar V, Sharma N, Shitiz K, Singh TR, Tandon C, Sood H, Chauhan RS (2015) An insight into conflux of metabolic traffic leading to picroside-I biosynthesis by tracking molecular time course changes in a medicinal herb, Picrorhiza kurroa. Plant Cell, Tissue Organ Cult 123:435–441

  • Kumar R, Kumar Y, Surender Singh G, Raj A (2016) Anti-inflammatory effect of Picrorhiza kurroa in experimental models of inflammation. Planta Med 82:1403–1409

    Article  CAS  PubMed  Google Scholar 

  • Kumar V, Bansal A, Rajinder SC (2017a) Modular design of picroside-II biosynthesis deciphered through NGS transcriptomes and metabolic intermediates analysis in naturally variant chemotypes of a medicinal herb, Picrorhiza kurroa. Front Plant Sci 8:564

    Article  PubMed  PubMed Central  Google Scholar 

  • Kumar V, Rajinder SC, Tandon T (2017b) Biosynthesis and therapeutic implications of iridoid glycosides from Picrorhiza genus: the road ahead. J Plant Biochem Biotechnol 26:1–13

    Article  CAS  Google Scholar 

  • Kumar V, Shitiz K, Rajinder SC, Sood H, Tandon C (2017c) Tracking dynamics of enzyme activities and their gene expression in Picrorhiza kurroa with respect to picroside accumulation. J Plant Biochem Biotechnol 25:125–132

    Article  CAS  Google Scholar 

  • Langer KM, Jones CR, Jaworski EA, Rushing GV, Kim JY, Clark DG, Colquhoun TA (2004) PhDAHP1 is required for floral volatile benzenoid/phenylpropanoid biosynthesis in Petunia × hybrid Cv ‘Mitchell Diploid.’ Phytochemistry 103:22–31

    Article  CAS  Google Scholar 

  • Lepelley M, Venkataramaiah M, McCarthy J, Rigoreau M, Crouzillat D, Chabrillange N, Alexandre Kochko A, Campa C (2012) Characterization, high-resolution mapping and differential expression of three homologous PAL genes in Coffea canephora (Rubiaceae). Planta 236:313–326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Y, Kim J, Pysh L, Chapple C (2015) Four isoforms of Arabidopsis 4-coumarate:CoA ligase have overlapping yet distinct roles in phenylpropanoid metabolism. Plant Physiol 169:2409–2421

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lois LM, Rodríguez-Concepción M, Gallego F, Campos N, Boronat A (2000) Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-d-xylulose 5-phosphate synthase. Plant J Cell Mol Biol 22:503–513

    Article  CAS  Google Scholar 

  • Lu S, Zhou L, Li L, Vincent LC (2006) Distinct roles of cinnamate 4-hydroxylase genes in Populus. Plant Cell Physiol 47:905–914

    Article  CAS  PubMed  Google Scholar 

  • Liu W, Zhang Z, Li W, Zhu W, Ren Z, Wang Z, Li L, Jia L, Zhu S, Ma Z (2018) Genome-wide identification and comparative analysis of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene family in Gossypium. Molecules 23:193

    Article  CAS  Google Scholar 

  • Moreira D, López-García P (2011) Paralogous gene. In: Gargaud M et al (eds) Encyclopedia of astrobiology. Springer, Berlin. https://doi.org/10.1007/978-3-642-11274-4_1732

    Chapter  Google Scholar 

  • Paetzold H, Garms S, Bartram S, Wieczorek J, Urós-Gracia EM, Rodríguez-Concepción M, Boland W, Strack D, Hause B, Walter MH (2010) The isogene 1-deoxy-d-xylulose 5-phosphate synthase 2 controls isoprenoid profiles, precursor pathway allocation, and density of tomato Trichomes. Mol Plant 3:904–916

    Article  CAS  PubMed  Google Scholar 

  • Panchy N, Lehti-Shiu M, Shiu SH (2016) Evolution of gene duplication in plants. Plant Physiol 171:2294–2316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pandit S, Shitiz K, Sood H, Chauhan RS (2012) Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa. J Plant Biochem Biotechnol 22:335–342

    Article  CAS  Google Scholar 

  • Pandit S, Shitiz K, Sood H, Kumar PN, Rajinder SC (2013) Expression pattern of fifteen genes of non-mevalonate (MEP) and mevalonate (MVA) pathways in different tissues of endangered medicinal herb Picrorhiza Kurroa with respect to picrosides content. Mol Biol Rep 40:1053–1563

    Article  CAS  PubMed  Google Scholar 

  • Rathee D, Thanki M, Bhuva S, Anandjiwala S, Agrawal R (2013) Iridoid glycosides-Kutkin, Picroside I, and Kutkoside from Picrorrhiza kurroa benth inhibits the invasion and migration of MCF-7 breast cancer cells through the down regulation of matrix metalloproteinases: 1st cancer update. Arab J Chem 6:49–58

    Article  CAS  Google Scholar 

  • Sharma N, Kumar V, Chauhan, RS (2016) Modulation of picroside-I biosynthesis in grown elicited shoots of picrorhiza kurroa in vitro. J Plant Growth Regul 35: 965–973

  • Shetty SN, Mengi S, Vaidya R, Vaidya AD (2010) A study of standardized extracts of Picrorhiza kurroa Royle Ex Benth in experimental nonalcoholic fatty liver disease. J Ayurveda Integr Med 1:203–210

    Article  PubMed  PubMed Central  Google Scholar 

  • Shitiz K, Sharma N, Pal T, Sood H, Chauhan RS (2015) NGS transcriptomes and enzyme inhibitors unravel complexity of picrosides biosynthesis in Picrorhiza kurroa Royle ex. Benth. PLoS ONE. https://doi.org/10.1371/journal.pone.0144546

    Article  PubMed  PubMed Central  Google Scholar 

  • Sood H, Chauhan RS (2010) Biosynthesis and accumulation of a medicinal compound, Picroside-I, in cultures of Picrorhiza kurroa Royle ex Benth. Plant Cell Tissue Organ Cult. https://doi.org/10.1007/s11240-009-9618-7

    Article  Google Scholar 

  • Suzuki M, Yukiko K, Noriko N, Hikaru S, Kiyoshi O, Hisashi K, Kazuo M (2004) Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels. Plant J 37:750–761

    Article  CAS  PubMed  Google Scholar 

  • Taylor JS, Raes J (2004) Duplication and divergence: the evolution of new genes and old ideas. Ann Rev Genet 38:615–643

    Article  CAS  PubMed  Google Scholar 

  • True JR, Carroll SB (2002) Gene co-option in physiological and morphological evolution. Annu Rev Cell Dev Biol 18:53–80

    Article  CAS  PubMed  Google Scholar 

  • Vashisht I, Mishra P, Pal T, Chanumolu S, Raj Singh T, Rajinder SC (2015) Mining NGS transcriptomes for miRNAs and dissecting their role in regulating growth, development, and secondary metabolites production in different organs of a medicinal herb, Picrorhiza Kurroa. Planta 241:1255–1268

    Article  CAS  PubMed  Google Scholar 

  • Vranová E, Coman D, Gruisse W (2013) Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol 64:665–700

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Guo B, Zhang F, Yao H, Miao Z, Kexuan T (2007) Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (CorylusAvellana L. Gasaway). J Biochem Mol Biol 40:861–869

    CAS  PubMed  Google Scholar 

  • Wang H, Zhao W, Choomuenwai V, Katherine T, Andrews Ronald JQ, Yunjiang F (2013) Chemical investigation of an antimalarial Chinese medicinal herb Picrorhiza scrophulariiflora. Bioorg Med Chem Lett 23:5915–5918

    Article  CAS  PubMed  Google Scholar 

  • Wei H, Movahedi A, Xu C, Sun W, Wang P, Li D, Zhuge Q (2020) Cross-talk between the methylerythritol phosphate and mevalonic acid pathways of isoprenoid biosynthesis in poplar. BioRxiv. https://doi.org/10.1101/2020.07.22.216804

    Article  PubMed  PubMed Central  Google Scholar 

  • Westfall CS, Xu A, Jez JM (2014) Structural evolution of differential amino acid effector regulation in plant chorismate mutases. J Biol Chem 289:28619–28628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xia J, Yajun L, Yao S, Li M, Zhu M, Huang K, Gao L, Xia T (2017) Characterization and expression profiling of Camellia sinensis cinnamate 4-hydroxylase genes in phenylpropanoid pathways. Genes 8:193

    Article  PubMed Central  CAS  Google Scholar 

  • Xiang B, Li X, Wang Y, Tian X, Yang Z, Ma L, Liu X, Wang Y (2017) Cloning and characterization of two iridoid synthase homologs from Swertia mussotii. Molecules 22:1387

    Article  PubMed Central  CAS  Google Scholar 

  • Xue L, Zilong H, Bi X, Wei X, Wei T, Wu S, Hu S (2019) Transcriptomic profiling reveals MEP pathway contributing to ginsenoside biosynthesis in Panax ginseng. BMC Genomics 20:383

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ye Z, Zhong R, Morrison W, Himmelsbach D (2001) Caffeoyl coenzyme A O-methyltransferase and lignin biosynthesis. Phytochemistry 57:1177–1185

    Article  CAS  PubMed  Google Scholar 

  • Zhang F, Liu W, Xia J, Zeng J, Xiang L, Zhu S, Zheng Q (2018) Molecular characterization of the 1-deoxy-d-xylulose 5-phosphate synthase gene family in Artemisia annua. Front Plant Sci 9:952

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India, for providing research grant in the form of program support on high-value medicinal plants to R. S. Chauhan. The authors are also highly thankful to Dr. Dipto Bhattacharyya for his critical readings and suggestions in the preparation of this manuscript. The support by Mr. Ashish Sharma, SRF is acknowledged for retrieving transcripts from NGS transcriptomes.

Funding

This work was financially supported by Department of Biotechnology, Ministry of Science and Technology, Government of India (Grant number: BT/COE/34/SP15268/2015) in the form of a Programme Support on high-value medicinal plants under Centres of Excellence.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Bennett University, Greater Noida, Uttar Pradesh, 201310, India

    Anjali Kharb & Rajinder S. Chauhan

Authors
  1. Anjali Kharb
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rajinder S. Chauhan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RSC and AK: Experiments designed; AK: Performed the experiments, Analysed the data, Manuscript preparation; RSC: Revised the manuscript critically and finally approved the manuscript to be published.

Corresponding author

Correspondence to Rajinder S. Chauhan.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

All the authors have consent for publication.

Additional information

Communicated by Stefan Hohmann.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 640 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kharb, A., Chauhan, R.S. Complexity of gene paralogues resolved in biosynthetic pathway of hepatoprotective iridoid glycosides in a medicinal herb, Picrorhiza kurroa through differential NGS transcriptomes. Mol Genet Genomics 296, 863–876 (2021). https://doi.org/10.1007/s00438-021-01787-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-021-01787-w

Keywords

Search

Navigation