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Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production

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Abstract

Echium plantagineum L. (Boraginaceae) is an invasive species in Australia and contains medicinal shikonins in its roots. In this study, the hairy root lines of E. plantagineum were established using Agrobacterium rhizogenes strain ATCC15834 and confirmed by the amplification of the rolB gene. Results showed significant difference in shikonin production between the hairy root lines in the 1/2B5 and M9 media. The biomass of the lines in the 1/2B5 medium was fivefold of that in the M9 medium. However, the components of detected shikonins were similar in these two liquid media. By contrast, different accumulation profiles appeared in the hairy root lines. HPLC analysis revealed the presence of nine possible related compounds, including shikonins, and acetylshikonin was the most abundant shikonin derivative. The content of acetylshikonin in the 1/2B5 medium (36.25 mg/L on average) was twofold of that in the M9 medium. Our results showed that the hairy root cultures of E. plantagineum can be used in enhancing the production of potential pharmaceutical compounds, such as acetylshikonin.

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References

  • Abd El-Mawla A (2010) Effect of certain elicitors on production of pyrrolizidine alkaloids in hairy root cultures of Echium rauwolfii. Pharmazie 65(3):224–226

    CAS  PubMed  Google Scholar 

  • Albreht A, Vovk I, Simonovska B, Srbinoska M (2009) Identification of shikonin and its ester derivatives from the roots of Echium italicum L. J Chromatogr A 1216(15):3156–3162

    CAS  PubMed  Google Scholar 

  • Babula P, Adam V, Havel L, Kizek R (2009) Noteworthy secondary metabolites naphthoquinones-their occurrence, pharmacological properties and analysis. Curr Pharm Anal 5(1):47–68

    CAS  Google Scholar 

  • Brigham LA, Michaels PJ, Flores HE (1999) Cell-specific production and antimicrobial activity of naphthoquinones in roots of Lithospermum erythrorhizon. Plant Physiol 119(2):417–428

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cequier-Sánchez E, Rodríguez C, Dorta-Guerra R, Ravelo ÁG, Zárate R (2011) Echium acanthocarpum hairy root cultures, a suitable system for polyunsaturated fatty acid studies and production. BMC Biotechnol 11(1):42

    PubMed  PubMed Central  Google Scholar 

  • Chacón J, Luebert F, Hilger HH, Ovchinnikova S, Selvi F, Cecchi L, Guilliams CM, Hasenstab-Lehman K, Sutorý K, Simpson MG (2016) The borage family (Boraginaceae s. str.): a revised infrafamilial classification based on new phylogenetic evidence, with emphasis on the placement of some enigmatic genera. Taxon 65(3):523–546

    Google Scholar 

  • Dresler S, Szymczak G, Wójcik M (2017) Comparison of some secondary metabolite content in the seventeen species of the Boraginaceae family. Pharm Biol 55(1):691–695

    CAS  PubMed  PubMed Central  Google Scholar 

  • Eruygur N (2018) A simple isocratic high-perfomance liquid chromatography method for the simultaneous determination of shikonin derivatives in some Echium species growing wild in Turkey. Turk J Pharm Sci 15(1):38–43

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fang R, Zou A, Zhao H, Wu F, Zhu Y, Zhao H, Liao Y, Tang R-J, Pang Y, Yang R (2016) Transgenic studies reveal the positive role of LeEIL-1 in regulating shikonin biosynthesis in Lithospermum erythrorhizon hairy roots. BMC Plant Biol 16(1):121

    PubMed  PubMed Central  Google Scholar 

  • Fujita Y, Hara Y, Ogino T, Suga C (1981a) Production of shikonin derivatives by cell suspension cultures of Lithospermum etythrorhizon. I. Effects of nitrogen sources on the production of shikonin derivatives. Plant Cell Rep 1:59–60

    CAS  PubMed  Google Scholar 

  • Fujita Y, Hara Y, Suga C, Morimoto T (1981b) Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. II. A new medium for the production of shikonin derivatives. Plant Cell Rep 1(2):61–63

    CAS  PubMed  Google Scholar 

  • Fujita Y, Maeda Y, Suga C, Morimoto T (1983) Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and Ko-shikon. Plant Cell Rep 2(4):192–193

    CAS  PubMed  Google Scholar 

  • Giri A, Narasu ML (2000) Transgenic hairy roots recent trends and applications. Biotechnol Adv 18(1):1–22

    CAS  PubMed  Google Scholar 

  • Grigulis K, Sheppard A, Ash J, Groves R (2001) The comparative demography of the pasture weed Echium plantagineum between its native and invaded ranges. J App Ecol 38(2):281–290

    Google Scholar 

  • Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006) Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol 9(3):341–346

    CAS  PubMed  Google Scholar 

  • Guo C, He J, Song X, Tan L, Wang M, Jiang P, Li Y, Cao Z, Peng C (2019) Pharmacological properties and derivatives of shikonin: a review in recent years. Pharmacol Res 2019:104463

    Google Scholar 

  • Ito Y, Onobori K, Yamazaki T, Kawamura Y (2011) Tigloylshikonin, a new minor shikonin derivative, from the roots and the commercial root extract of Lithospermum erythrorhizon. Chem Pharm Bull 59(1):117–119

    CAS  PubMed  Google Scholar 

  • Katelaris C, Baldo B, Howden M, Matthews P, Walls R (1982) Investigation of the involvement of Echium plantagineum (Paterson’s curse) in seasonal allergy: IgE antibodies to Echium and other weed pollens. Allergy 37(1):21–28

    CAS  PubMed  Google Scholar 

  • Khazaei A, Bahramnejad B, Mozafari A-A, Dastan D, Mohammadi S (2019) Hairy root induction and Farnesiferol B production of endemic medicinal plant Ferula pseudalliacea. 3 Biotech 9(11):407

    PubMed  Google Scholar 

  • Kim Y, Wyslouzil BE, Weathers PJ (2002) Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cell Dev Biol-Plant 38(1):1–10

    CAS  Google Scholar 

  • Malik S, Bhushan S, Sharma M, Ahuja PS (2016) Biotechnological approaches to the production of shikonins: a critical review with recent updates. Crit Rev Biotechnol 36(2):327–340

    CAS  PubMed  Google Scholar 

  • Mizukami H, Konoshima M, Tabata M (1977) Effect of nutritional factors on shikonin derivative formation in Lithospermum callus cultures. Phytochemistry 16(8):1183–1186

    CAS  Google Scholar 

  • Papageorgiou V, Assimopoulou A, Ballis A (2008) Alkannins and shikonins: a new class of wound healing agents. Curr Med Chem 15(30):3248–3267

    CAS  PubMed  Google Scholar 

  • Pietrosiuk A, Sykłowska-Baranek K, Wiedenfeld H, Wolinowska R, Furmanowa M, Jaroszyk E (2006) The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm. Plant Cell Rep 25(10):1052–1058

    CAS  PubMed  Google Scholar 

  • Piggin C (1982) The biology of Australian weeds. 8. Echium plantagineum L. J Austral Inst Agric Sci 48:3–16

    Google Scholar 

  • Qi J-L, Zhang W-J, Liu S-H, Wang H, Sun D-Y, Xu G-H, Shi M-W, Liu Z, Zhang M-S, Zhang H-M (2008) Expression analysis of light-regulated genes isolated from a full-length-enriched cDNA library of Onosma paniculatum cell cultures. J Plant Physiol 165(14):1474–1482

    CAS  PubMed  Google Scholar 

  • Renouard S, Corbin C, Drouet S, Medvedec B, Doussot J, Colas C, Maunit B, Bhambra A, Gontier E, Jullian N (2018) Investigation of Linum flavum (L.) hairy root cultures for the production of anticancer aryltetralin lignans. Int J Mol Sci 19(4):990

    PubMed Central  Google Scholar 

  • Sathyanarayana B, Verghese DB (2007) Plant tissue culture: practices and new experimental protocols. I. K. International Publishing House, New Delhi

    Google Scholar 

  • Shcherbanovskii L, Luks YA (1974) Shikonin from Echium lycopsis. Chem Nat Compd 10(4):517–517

    Google Scholar 

  • Singh RS, Gara RK, Bhardwaj PK, Kaachra A, Malik S, Kumar R, Sharma M, Ahuja PS, Kumar S (2010) Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston]. BMC Mol Biol 11(1):88

    PubMed  PubMed Central  Google Scholar 

  • Skoneczny D, Weston P, Zhu X, Gurr G, Callaway R, Barrow R, Weston L (2017) Metabolic profiling and identification of shikonins in root periderm of two invasive Echium spp. weeds in Australia. Molecules 22(2):330

    PubMed Central  Google Scholar 

  • Skoneczny D, Zhu X, Weston PA, Gurr GM, Callaway RM, Weston LA (2019) Production of pyrrolizidine alkaloids and shikonins in Echium plantagineum L. in response to various plant stressors. Pest Manag Sci 75(9):2530–2541

    CAS  PubMed  Google Scholar 

  • Srivastava S, Srivastava AK (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol 27(1):29–43

    CAS  PubMed  Google Scholar 

  • Sykłowska-Baranek K, Pietrosiuk A, Gawron A, Kawiak A, Łojkowska E, Jeziorek M, Chinou I (2012) Enhanced production of antitumour naphthoquinones in transgenic hairy root lines of Lithospermum canescens. Plant Cell Tissue Organ Cult 108(2):213–219

    Google Scholar 

  • Tatsumi K, Yano M, Kaminade K, Sugiyama A, Sato M, Toyooka K, Aoyama T, Sato F, Yazaki K (2016) Characterization of shikonin derivative secretion in Lithospermum erythrorhizon hairy roots as a model of lipid-soluble metabolite secretion from plants. Front Plant Sci 7:1066

    PubMed  PubMed Central  Google Scholar 

  • Tsukada M, Tabata M (1984) Intracellular localization and secretion of naphthoquinone pigments in cell cultures of Lithospermum erythrorhizon. Planta Med 50(04):338–341

    CAS  PubMed  Google Scholar 

  • Verpoorte R, Alfermann AW (2000) Metabolic engineering of plant secondary metabolism. Springer, Dordrecht

    Google Scholar 

  • Weston LA, Weston PA, McCully M (2011) Production of bioactive napthoquinones by roots of Paterson’s curse (Echium plantagineum)—Implications for invasion success. Pak J Weed Sci Res 18:677–686

    Google Scholar 

  • Wu F-Y, Tang C-Y, Guo Y-M, Bian Z-W, Fu J-Y, Lu G-H, Qi J-L, Pang Y-J, Yang Y-H (2017) Transcriptome analysis explores genes related to shikonin biosynthesis in Lithospermeae plants and provides insights into Boraginales’ evolutionary history. Sci Rep 7(1):4477

    PubMed  PubMed Central  Google Scholar 

  • Yazaki K (2004) Natural products and metabolites. In: Christou P and Klee H (ed) Handbook of plant biotechnology. Doi: 10.1002/0470869143.kc039

  • Yazaki K (2017) Lithospermum erythrorhizon cell cultures: present and future aspects. Plant Biotechnol 34(3):131–142

    CAS  Google Scholar 

  • Zárate R, Cequier-Sánchez E, Rodríguez C, Dorta-Guerra R, El Jaber-Vazdekis N, Ravelo ÁG (2013) Improvement of polyunsaturated fatty acid production in Echium acanthocarpum transformed hairy root cultures by application of different abiotic stress conditions. ISRN Biotechnol 2013:169510

    PubMed  PubMed Central  Google Scholar 

  • Zárate R, Cequier-Sánchez E, El Jaber-Vazdekis N, Rodríguez C, Dorta-Guerra R, Ravelo ÁG (2016) Healthy omega-3 enhancement in Echium acanthocarpum transformed hairy roots by overexpression of a 6-desaturase gene from Primula vialli. SCIREA J Bio 1:1–40

    Google Scholar 

  • Zare K, Nazemiyeh H, Movafeghi A, Khosrowshahli M, Motallebi-Azar A, Dadpour M, Omidi Y (2010) Bioprocess engineering of Echium italicum L.: induction of shikonin and alkannin derivatives by two-liquid-phase suspension cultures. Plant Cell Tissue Organ Cult 100(2):157–164

    CAS  Google Scholar 

  • Zare K, Khosrowshahli M, Nazemiyeh H, Movafeghi A, Azar AM, Omidi Y (2011) Callus culture of Echium italicum L. towards production of a shikonin derivative. Nat Prod Res 25(16):1480–1487

    CAS  PubMed  Google Scholar 

  • Zhao H, Chang Q, Zhang D, Fang R, Wu F, Wang X, Lu G, Qi J, Yang Y (2015) Overexpression of LeMYB1 enhances shikonin formation by up-regulating key shikonin biosynthesis-related genes in Lithospermum erythrorhizon. Biol Plantarum 59(3):429–435

    CAS  Google Scholar 

  • Zhu X, Skoneczny D, Weidenhamer JD, Mwendwa JM, Weston PA, Gurr GM, Callaway RM, Weston LA (2016) Identification and localization of bioactive naphthoquinones in the roots and rhizosphere of Paterson's curse (Echium plantagineum), a noxious invader. J Exp Bot 67(12):3777–3788

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (U1903201, 31970321, 31771413, 31670298), the Open Project Program from MOE Key Laboratory of Molecular Epigenetics of China, and the Program for Changjiang Scholars and Innovative Research Team in University from the Ministry of Education of China (IRT_14R27).

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YHY, JLQ, and GHL conceived and designed the experiments. JYF, HZ, and JXB performed the experiments. JYF, HZ, ZLW, and RJF analyzed the data. AF, MKY, BL, TMY, and YJP contributed to resources. JYF and HZ wrote the draft of manuscript. YHY, JLQ, and GHL contributed to review and edit the manuscript. All the authors carefully checked and approved the final manuscript.

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Correspondence to Gui-Hua Lu, Jin-Liang Qi or Yong-Hua Yang.

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Fu, JY., Zhao, H., Bao, JX. et al. Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production. 3 Biotech 10, 429 (2020). https://doi.org/10.1007/s13205-020-02419-7

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