Skip to main content
SpringerLink
Log in

Two light responsive WRKY genes exhibit positive and negative correlation with picroside content in Picrorhiza kurrooa Royle ex Benth, an endangered medicinal herb

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

Picrorhiza kurrooa is an endangered herb known to produce the medicinally important picrosides through isoprenoid pathway. The present work showed the functionality of WRKY motifs (TGAC cis-acting elements) present in the promoters of regulatory genes 3-hydroxy-3-methylglutaryl coenzyme A reductase (Pkhmgr) and 1-deoxy-d-xylulose-5-phosphate synthase (Pkdxs) of the picrosides biosynthetic pathway by electrophoretic mobility shift assay. Also, the two WRKY genes, PkdWRKY and PksWRKY, were characterized and found to contain double and single characteristic WRKY domains, respectively along with a zinc-finger motif in each domain. Expression analysis revealed that PkdWRKY and PksWRKY exhibited a positive and negative correlation, respectively, with picrosides content under the environment of light and in different tissues. Functional evaluation in yeast showed DNA binding ability of both PksWRKY and PkdWRKY; however, only PkdWRKY exhibited transcriptional activation ability. Transient overexpression of PkdWRKY and PksWRKY in tobacco modulated the expression of selected native genes of tobacco involved in MVA and MEP pathway suggesting functionality of PkdWRKY and PksWRKY in planta. Collectively, data suggested that PkdWRKY and PksWRKY might be positive and negative regulators, respectively in the picrosides biosynthetic pathway.

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

Similar content being viewed by others

Abbreviations

MVA:

Mevalonate

MEP:

Methylerythritol phosphate

EMSA:

Electrophoretic mobility shift assay

NP:

Nucleo-protein

TF:

Transcription factor

References

  • Akagi A, Fukushima S, Okada K, Jiang CJ, Yoshida R, Nakayama A et al (2014) WRKY45 dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction. Plant Mol Biol 86:171–183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 1(5):2320–2325

    Article  CAS  PubMed  Google Scholar 

  • Berg JM, Shi Y (1996) The galvanization of biology: a growing appreciation for the roles of zinc. Science 271:1081–1085

    Article  CAS  PubMed  Google Scholar 

  • Calkhoven CF, Geert AB (1996) Multiple steps in the regulation of transcription factor level and activity. Biochem J 317:329–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dai X, Wang Y, Zhang WH (2016) OsWRKY74, a WRKY transcription factor, modulates tolerance to phosphate starvation in rice. J Exp Bot 67:947–960

    Article  CAS  PubMed  Google Scholar 

  • Dang FF, Wang YN, She JJ, Lei YF, Liu ZQ, Eulgem T et al (2014) Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection. Physiol Plant 150:397–411

    Article  CAS  PubMed  Google Scholar 

  • Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206

    Article  CAS  PubMed  Google Scholar 

  • Fan ZQ, Tan XL, Shan W, Kuang JF, Lu WJ, Chen JY (2017) BrWRKY65, a WRKY transcription factor, is involved in regulating three leaf senescence-associated genes in Chinese flowering cabbage. Int J Mol Sci 18:1228

    Article  CAS  PubMed Central  Google Scholar 

  • Gahlan P, Singh HR, Shankar R, Sharma N, Kumari A, Chawla V et al (2012) De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments. BMC Genom 13:126

    Article  CAS  Google Scholar 

  • Gaudinier A, Tang M, Kliebenstein DJ (2015) Transcriptional networks governing plant metabolism. Curr Plant Biol 3–4:56–64

    Article  Google Scholar 

  • Ghawana S, Paul A, Kumar H, Kumar A, Singh H, Bhardwaj PK et al (2011) An RNA isolation system for plant tissues rich in secondary metabolites. BMC Res Notes 4:85

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Han J, Wang H, Lundgren A, Brodelius PE (2014) Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants. Phytochemistry 102:89–96

    Article  CAS  PubMed  Google Scholar 

  • Hara K, Yagi M, Kusano T, Sano H (2000) Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding. Mol Gen Genet 263:30–37

    Article  CAS  PubMed  Google Scholar 

  • Husain GM, Singh PN, Kumar V (2009) Antidiabetic activity of standardized extract of Picrorhiza kurroa in rat model of NIDDM. Drug Discov Ther 3:88–92

    CAS  PubMed  Google Scholar 

  • Jiang W, Yu D (2009) Arabidopsis WRKY2 transcription factor mediates seed germination and post-germination arrest of development by abscisic acid. BMC Plant Biol 9:96

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kawauchi M, Arima TH, Kuroyanagi M (2016) Molecular cloning and transcriptional analysis of WRKY and solavetivone biosynthetic genes in the hairy roots of Hyoscyamus albus. Plant Gene 5:78–86

    Article  CAS  Google Scholar 

  • Kawoosa T, Singh H, Kumar A, Sharma SK, Devi K, Dutt S et al (2010) Light and temperature regulated terpene biosynthesis: hepatoprotective monoterpene picroside accumulation in Picrorhiza kurrooa. Funct Integr Genom 10:393–404

    Article  CAS  Google Scholar 

  • Klok EJ, Wilson IW, Wilson D, Chapman SC, Ewing RM, Somerville SC et al (2002) Expression profile analysis of the low-oxygen response in Arabidopsis root cultures. Plant Cell 14:2481–2494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lacomme C, Chapman S (2008) Use of potato virus X (PVX)–based vectors for gene expression and virus induced gene silencing (VIGS). Curr Protoc Microbiol 8(1):16I–I21

    Article  Google Scholar 

  • Lagace M, Matton DP (2004) Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta 219:185–189

    Article  CAS  PubMed  Google Scholar 

  • Li HL, Guo D, Yang ZP, Tang X, Peng SQ (2014) Genome-wide identification and characterization of WRKY gene family in Hevea brasiliensis. Genomics 104:14–23

    Article  CAS  PubMed  Google Scholar 

  • Li P, Matsunaga K, Yamakuni T, Ohizumi Y (2000) Potentiation of nerve growth factor-action by picrosides I and II, natural iridoids, in PC12D cells. Eur J Pharmacol 406:203–208

    Article  CAS  PubMed  Google Scholar 

  • Li P, Matsunaga K, Yamakuni T, Ohizumi Y (2002) Picrosides I and II, selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells. Life Sci 71:1821–1835

    Article  CAS  PubMed  Google Scholar 

  • Li S, Fu Q, Chen L, Huang W, Yu D (2011) Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta 233:1237–1252

    Article  CAS  PubMed  Google Scholar 

  • Li S, Zhang P, Zhang M, Fu C, Yu L (2013) Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis. Plant Biol 15:19–26

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Long L, Shaohua M, Zhanchao C, Yuanwen C, Ying Z, Ying M et al (2017) Characterization and expression analysis of the WRKY gene family in moso bamboo. Sci Rep 7:6675

    Article  CAS  Google Scholar 

  • Ma D, Pu G, Lei C, Ma L, Wang H, Guo Y et al (2009) Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis. Plant Cell Physiol 50:2146–2161

    Article  CAS  PubMed  Google Scholar 

  • Nandave M, Ojha SK, Kumari S, Nag TC, Mehra R, Narang R et al (2013) Cardioprotective effect of root extract of Picrorhiza kurroa (Royle Ex Benth) against isoproterenol-induced cardiotoxicity in rats. Indian J Exp Biol 51:694–701

    PubMed  Google Scholar 

  • Park CJ, Shin YC, Lee BJ, Kim KJ, Kim JK, Paek KH (2006) A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to tobacco mosaic virus and Xanthomonas campestris. Planta 223:168–179

    Article  CAS  PubMed  Google Scholar 

  • Phukan UJ, Jeena GS, Shukla RK (2016) WRKY transcription factors: molecular regulation and stress responses in plants. Front Plant Sci 7:760

    Article  PubMed  PubMed Central  Google Scholar 

  • Printen JA, Sprague GFJ (1994) Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics 138:609–619

    CAS  PubMed  PubMed Central  Google Scholar 

  • Puri A, Saxena RP, Sumati GPY, Kulshreshtha DK, Saxena KC, Dhawan BN (1992) Immunostimulant activity of Picroliv, the iridoid glycoside fraction of Picrorhiza kurrooa, and its protective action against Leishmania donovani infection in hamsters. Planta Med 58:528–532

    Article  CAS  PubMed  Google Scholar 

  • Rastogi R, Srivastava AK, Rastogi K (2001) Long term effect of aflatoxin B1 on lipid peroxidation in rat liver and kidney: effect of picroliv and silymarin. Phytotherapy Res 15:307–310

    Article  CAS  Google Scholar 

  • Rohmer M (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat Prod Rep 16:565–574

    Article  CAS  PubMed  Google Scholar 

  • Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15:247–258

    Article  CAS  PubMed  Google Scholar 

  • Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15:5690–5700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Saraswat B, Visen PK, Patnaik GK, Dhawan BN (1993) Anticholestatic effect of picroliv, active hepatoprotective principle of Picrorhiza kurrooa, against carbon tetrachloride induced cholestasis. Indian J Exp Biol 31(4):316–318

    CAS  PubMed  Google Scholar 

  • Schluttenhofer C, Yuan L (2015) Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol 167:295–306

    Article  CAS  PubMed  Google Scholar 

  • Shang Y, Yan L, Liu Z, Cao Z, Mei C, Xin Q et al (2010) The Mg-chelatase H subunit of Arabidopsis antagonizes a group of transcription repressors to relieve ABA-responsive genes of inhibition. Plant Cell 22:1909–1935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sinha S, Bhat J, Joshi M, Sinkar V, Ghaskadbi S (2011) Hepatoprotective activity of Picrorhiza kurroa Royle Ex. Benth extract against alcohol cytotoxicity in mouse liver slice culture. Int J Green Pharmacy 5(3):244–253

    Article  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Venkataraman K (2009) India’s Biodiversity Act 2002 and its role in conservation. Trop Ecol 50(1):23–30

    Google Scholar 

  • Vom Endt D, Kijne JW, Memelink J (2002) Transcription factors controlling plant secondary metabolism: what regulates the regulators? Phytochemistry 61:107–114

    Article  Google Scholar 

  • Wang JJ, Tao F, An F, Zou YP, Tian W, Chen XM et al (2017) Wheat transcription factor TaWRKY70 is positively involved in high-temperature seedling plant resistance to Puccinia striiformis f. sp. tritici. Mol Plant Pathol 18:649–661

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Guo D, Li HL, Peng SQ (2013) Characterization of HbWRKY1, a WRKY transcription factor from Hevea brasiliensis that negatively regulates HbSRPP. Plant Physiol Biochem 71:283–289

    Article  CAS  PubMed  Google Scholar 

  • Weigel D, Glazebrook J (2006) Transformation of agrobacterium using the freeze-thaw method. CSH protocols 7:1031–1036

    Google Scholar 

  • Wu HL, Ni ZF, Yao YY, Guo GG, Sun QX (2008) Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.). Prog Nat Sci 18:697–705

    Article  CAS  Google Scholar 

  • Wu KL, Guo ZJ, Wang HH, Li J (2005) The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res 12:9–26

    Article  CAS  PubMed  Google Scholar 

  • Xu YH, Wang JW, Wang S, Wang JY, Chen XY (2004) Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A. Plant Physiol 135:507–515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Q, Zhu J, Ni Y, Cai Y, Zhang Z (2012) Expression profiling of HbWRKY1, an ethephon-induced WRKY gene in latex from Hevea brasiliensis in responding to wounding and drought. Trees (Berl) 26:587–595

    Article  CAS  Google Scholar 

  • Zhang ZL, Shin M, Zou X, Huang J, Ho TH, Shen Q (2009) A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells. Plant Mol Biol 70:139–151

    Article  CAS  PubMed  Google Scholar 

  • Zhang ZL, Xie Z, Zou XL, Casaretto J, Ho THD, Shen QXJ (2004) A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol 134:1500–1513

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng Z, Mosher SL, Fan B, Klessig DF, Chen Z (2007) Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae. BMC Plant Biol 7:2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

TS is thankful to Indian Council of Medical Research (ICMR), for junior/ senior research fellowship. The authors acknowledge financial support from the Council of Scientific and Industrial Research (CSIR), India for funding projects entitled “Genomics of medicinal plants and agronomically important traits (PlaGen), BSC0107” and “Plant diversity: studying adaptation biology and understanding/exploiting medicinally important plants for useful bioactives, SIMPLE-BSC0109”. The manuscript represents CSIR-IHBT communication no. 4232.

Author information

Author notes

  1. Tanvi Sharma and Tabasum Kawoosa contributed equally to this work.

Authors and Affiliations

  1. Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, P.O. Box No. 6, Palampur, Himachal Pradesh, 176061, India

    Tanvi Sharma, Tabasum Kawoosa, Parul Gahlan, Damini Sharma, Anish Kaachra, Vipin Hallan & Sanjay Kumar

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, 201002, India

    Tanvi Sharma & Sanjay Kumar

Authors
  1. Tanvi Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tabasum Kawoosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Parul Gahlan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Damini Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anish Kaachra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vipin Hallan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sanjay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

TS performed EMSA, gene expression analysis, yeast hybrid assays and wrote the manuscript. TK performed cloning and in silico analysis and wrote the first draft of manuscript. PG performed picroside estimation. DS performed agro-infilteration in tobacco and isolated RNA and DNA from infilterated leaves. AK prepared transient overexpression constructs and performed PCR and qRT-PCR analysis. VH supervised yeast hybrid experiments and finalised the manuscript. SK conceived the study, designed the experiments, coordinated the study and finalised the manuscript. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Sanjay Kumar.

Ethics declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 891 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sharma, T., Kawoosa, T., Gahlan, P. et al. Two light responsive WRKY genes exhibit positive and negative correlation with picroside content in Picrorhiza kurrooa Royle ex Benth, an endangered medicinal herb. 3 Biotech 10, 255 (2020). https://doi.org/10.1007/s13205-020-02249-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-020-02249-7

Keywords

Search

Navigation