Skip to main content
SpringerLink
Log in

Overexpressing fusion proteins of 4-coumaroyl-CoA ligase (4CL) and stilbene synthase (STS) in tobacco plants leading to resveratrol accumulation and improved stress tolerance

  • Original Article
  • Published:
Plant Biotechnology Reports Aims and scope Submit manuscript

Abstract

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a phytoalexin produced in plants in response to pathogen attack as a part of plant defense response and it is also a highly bioactive substance of pharmaceutical interest. To obtain transgenic plants with a high level of resveratrol, two enzymes in the last two steps of resveratrol synthesis, 4-coumaroyl-CoA ligase (4CL) and stilbene synthase (STS), were fused together by a glycine–serine–glycine (GSG) tripeptide linker, and the 4CL-GSG-STS construct driven by a CaMV35S promoter was transformed into tobacco (Nicotiana benthamiana) by Agrobacterium-mediated method. In the transgenic plants, a high resveratrol level was detected (21.05 µg/g fresh weight) by high-pressure liquid chromatography (HPLC), which is higher than previous transgenic plants with only STS gene overexpression. In addition to resistance to pathogen, transgenic plants showed improved tolerance to salt and osmotic stresses, and the lower level of malondialdehyde (MDA) in transgenic plants suggested that resveratrol could protect plant membrane lipid from peroxidation under abiotic stresses.

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

Similar content being viewed by others

References

  • Aleynova OA, Grigorchuk VP, Dubrovina AS, Rybin VG, Kiselev KV (2016) Stilbene accumulation in cell cultures of Vitis amurensis Rupr. overexpressing VaSTS1. VaSTS2, and VaSTS7 genes. Plant Cell Tiss Org 125:329–339

    Article  CAS  Google Scholar 

  • Bell MR, Engleka MJ, Malik A, Strickler JE (2013) To fuse or not to fuse: what is your purpose? Protein Sci 22:1466–1477

    Article  CAS  Google Scholar 

  • Bostanghadiri N, Pormohammad A, Chirani AS, Pouriran R, Erfanimanesh S, Hashemi A (2017) Comprehensive review on the antimicrobial potency of the plant polyphenol resveratrol. Biomed Pharmacother 95:1588–1595

    Article  CAS  Google Scholar 

  • Bulow L (1990) Preparation of artificial bifunctional enzymes by gene fusion. Biochem Soc Symp 57:123–133

    CAS  PubMed  Google Scholar 

  • Cheng S, Xie X, Xu Y, Zhang C, Wang X, Zhang J, Wang Y (2016) Genetic transformation of a fruit-specific, highly expressed stilbene synthase gene from Chinese wild Vitis quinquangularis. Planta 243:1041–1053

    Article  CAS  Google Scholar 

  • Chong J, Poutaraud A, Hugueney P (2009) Metabolism and roles of stilbenes in plants. Plant Sci 177:143–155

    Article  CAS  Google Scholar 

  • Chu M, Pedreno MA, Alburquerque N, Faize L, Burgos L, Almagro L (2017) A new strategy to enhance the biosynthesis of trans-resveratrol by overexpressing stilbene synthase gene in elicited Vitis vinifera cell cultures. Plant Physiol Biochem 113:141–148

    Article  CAS  Google Scholar 

  • Coutos-Thevenot P et al (2001) In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR 10 promoter. J Exp Bot 52:901–910

    Article  CAS  Google Scholar 

  • Dubrovina AS, Kiselev KV (2017) Regulation of stilbene biosynthesis in plants. Planta 246:597–623

    Article  CAS  Google Scholar 

  • Fan C, Pu N, Wang X, Wang Y, Fang L, Xu W, Zhang J (2008) Agrobacterium-mediated genetic transformation of grapevine (Vitis vinifera L.) with a novel stilbene synthase gene from Chinese wild Vitis pseudoreticulata. Plant Cell Tiss Org 92:197–206

    Article  CAS  Google Scholar 

  • Guo H et al (2017) Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase. Mol Biosyst 13:598–606

    Article  CAS  Google Scholar 

  • Hain R et al (1993) Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 361:153–156

    Article  CAS  Google Scholar 

  • Hasan M, Bae H (2017) An overview of stress-induced resveratrol synthesis in grapes: perspectives for resveratrol-enriched grape products. Molecules 22:294

    Article  Google Scholar 

  • Hatmi S, Trotel-Aziz P, Villaume S, Couderchet M, Clement C, Aziz A (2014) Osmotic stress-induced polyamine oxidation mediates defence responses and reduces stress-enhanced grapevine susceptibility to Botrytis cinerea. J Exp Bot 65:75–88

    Article  CAS  Google Scholar 

  • Hipskind JD, Paiva NL (2000) Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Mol Plant Microbe Interact 13:551–562

    Article  CAS  Google Scholar 

  • Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231

    Article  CAS  Google Scholar 

  • Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68

    Article  CAS  Google Scholar 

  • Howitz KT et al (2003) Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425:191–196

    Article  CAS  Google Scholar 

  • Jeandet P et al (2010) Biosynthesis, metabolism, molecular engineering, and biological functions of stilbene phytoalexins in plants. Biofactors 36:331–341

    Article  CAS  Google Scholar 

  • Jeong YJ et al (2016) Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway. Plant Mol Biol 92:117–129

    Article  CAS  Google Scholar 

  • Kato M, Shimizu S (1987) Chlorophyll metabolism in higher plants. VII. Chlorophyll degradation in senescing tobacco leaves; phenolic-dependent peroxidative degradation. Can J Bot 65:729–735

    Article  CAS  Google Scholar 

  • Kursvietiene L, Staneviciene I, Mongirdiene A, Bernatoniene J (2016) Multiplicity of effects and health benefits of resveratrol. Medicina (Mex) 52:148–155

    Article  Google Scholar 

  • Lekli I, Ray D, Das DK (2010) Longevity nutrients resveratrol, wines and grapes. Genes Nutr 5:55–60

    Article  CAS  Google Scholar 

  • Li M, Kildegaard KR, Chen Y, Rodriguez A, Borodina I, Nielsen J (2015) De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae. Metab Eng 32:1–11

    Article  Google Scholar 

  • Lim JD, Yun SJ, Chung IM, Yu CY (2005) Resveratrol synthase transgene expression and accumulation of resveratrol glycoside in Rehmannia glutinosa. Mol Breed 16:219–233

    Article  CAS  Google Scholar 

  • Liu Y et al (2010) Characterization of a DRE-binding transcription factor from asparagus (Asparagus officinalis L.), and its overexpression in Arabidopsis resulting in salt and drought-resistant transgenic plants. Int J Plant Sci 171:12–23

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Lu Y, Shao D, Shi J, Huang Q, Yang H, Jin M (2016) Strategies for enhancing resveratrol production and the expression of pathway enzymes. Appl Microbiol Biotechnol 100:7407–7421

    Article  CAS  Google Scholar 

  • Luo Z, Guo H, Yang Y, Yang M, Ma L, Wang Y (2015) Heterologous overexpression of resveratrol synthase (PcPKS5) gene enhances antifungal and mite aversion by resveratrol accumulation. Eur J Plant Pathol 142:547–556

    Article  CAS  Google Scholar 

  • Ma BG, Duan XY, Niu JX, Ma C, Hao QN, Zhang LX, Zhang HP (2009) Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker. Biotechnol Lett 31:163–169

    Article  CAS  Google Scholar 

  • Nandagopal K, Halder M, Dash B, Nayak S, Jha S (2017) Biotechnological approaches for production of anti-cancerous compounds resveratrol, podophyllotoxin and zerumbone. Curr Med Chem. https://doi.org/10.2174/0929867324666170404145656

    Article  PubMed  Google Scholar 

  • Nguyen C, Savouret JF, Widerak M, Corvol MT, Rannou F (2017) Resveratrol, potential therapeutic interest in joint disorders: a critical narrative review. Nutrients. https://doi.org/10.3390/nu9010045

    Article  PubMed  PubMed Central  Google Scholar 

  • Punja ZK (2001) Genetic engineering of plants to enhance resistance to fungal pathogens—a review of progress and future prospects. Can J Plant Pathol 23:216–235

    Article  CAS  Google Scholar 

  • Riviere C, Pawlus AD, Merillon JM (2012) Natural stilbenoids: distribution in the plant kingdom and chemotaxonomic interest in Vitaceae. Nat Prod Rep 29:1317–1333

    Article  CAS  Google Scholar 

  • Sakharov IY, Ardila GB (1999) Variations of peroxidase activity in cocoa (Theobroma cacao L.) beans during their ripening, fermentation and drying. Food Chem 65:51–54

    Article  CAS  Google Scholar 

  • Schroder J, Schroder G (1990) Stilbene and chalcone synthases: related enzymes with key functions in plant-specific pathways. Z Naturforsch C 45:1–8

    Article  CAS  Google Scholar 

  • Sebai H, Gadacha W, Sani M, Aouani E, Ghanem-Boughanmi N, Ben-Attia M (2009) Protective effect of resveratrol against lipopolysaccharide-induced oxidative stress in rat brain. Brain Inj 23:1089–1094

    Article  Google Scholar 

  • Shin SY, Han NS, Park YC, Kim MD, Seo JH (2011) Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes. Enzyme Microb Technol 48:48–53

    Article  CAS  Google Scholar 

  • Sinnhuber RO, Chang YT (2006) Characterization of the red pigment in the Z-thiobarbituric acid determination of oxidative rancidity. J Food Sci 23:626–634

    Article  Google Scholar 

  • Tantong S, Incharoensakdi A, Sirikantaramas S, Lindblad P (2016) Potential of Synechocystis PCC 6803 as a novel cyanobacterial chassis for heterologous expression of enzymes in the trans-resveratrol biosynthetic pathway. Protein Expr Purif 121:163–168

    Article  CAS  Google Scholar 

  • Theodotou M, Fokianos K, Mouzouridou A, Konstantinou C, Aristotelous A, Prodromou D, Chrysikou A (2017) The effect of resveratrol on hypertension: a clinical trial. Exp Ther Med 13:295–301

    Article  CAS  Google Scholar 

  • Thiel G, Rossler OG (2017) Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors. Pharmacol Res 117:166–176

    Article  CAS  Google Scholar 

  • Titarenko E, Lopez-Solanilla E, Garcia-Olmedo F, Rodriguez-Palenzuela P (1997) Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco. J Bacteriol 179:6699–6704

    Article  CAS  Google Scholar 

  • Truong VL, Jun M, Jeong WS (2018) Role of resveratrol in regulation of cellular defense systems against oxidative stress. Biofactors 44:36–49

    Article  CAS  Google Scholar 

  • Wang Y, Yu O (2012) Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells. J Biotechnol 157:258–260

    Article  CAS  Google Scholar 

  • Wang Y, Yi H, Wang M, Yu O, Jez JM (2011) Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase. J Am Chem Soc 133:20684–20687

    Article  CAS  Google Scholar 

  • Wang C et al (2017) Characterization of stilbene synthase genes in mulberry (Morus atropurpurea) and metabolic engineering for the production of resveratrol in Escherichia coli. J Agric Food Chem 65:1659–1668

    Article  CAS  Google Scholar 

  • Watts KT, Lee PC, Schmidt-Dannert C (2006) Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli. BMC Biotechnol 6:22

    Article  Google Scholar 

  • Weiskirchen S, Weiskirchen R (2016) Resveratrol: how much wine do you have to drink to stay healthy? Advances in nutrition (Bethesda. Md) 7:706–718

    CAS  Google Scholar 

  • Zhang Y et al (2006) Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. J Am Chem Soc 128:13030–13031

    Article  CAS  Google Scholar 

  • Zheng S et al (2015) Evaluating the effect of expressing a peanut resveratrol synthase gene in rice. PLoS One 10:e0136013

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31370674; 21606020); Beijing Natural Science Foundation (2164059).

Author information

Authors and Affiliations

  1. Key Laboratory of Urban Agriculture (North China) Ministry of Agriculture, Beijing University of Agriculture, Beijing, 102206, China

    Xuancheng He, Feiyan Xue, Lulu Zhang, Huili Guo, Lanqing Ma & Mingfeng Yang

Authors
  1. Xuancheng He
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feiyan Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lulu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Huili Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lanqing Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mingfeng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HX, XF, ZL and GH conducted experiments; YMF and MLQ designed research and wrote the main manuscript text; all authors read and approved the manuscript.

Corresponding authors

Correspondence to Lanqing Ma or Mingfeng Yang.

Ethics declarations

Conflict of interest

The authors declare no competing financial interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1410 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

He, X., Xue, F., Zhang, L. et al. Overexpressing fusion proteins of 4-coumaroyl-CoA ligase (4CL) and stilbene synthase (STS) in tobacco plants leading to resveratrol accumulation and improved stress tolerance. Plant Biotechnol Rep 12, 295–302 (2018). https://doi.org/10.1007/s11816-018-0494-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11816-018-0494-7

Keywords

Search

Navigation