Frontiers of Chemical Science and Engineering

, Volume 11, Issue 4, pp 521–528 | Cite as

Gene delivery into isolated Arabidopsis thaliana protoplasts and intact leaves using cationic, α-helical polypeptide

Research Article


The application of gene delivery materials has been mainly focused on mammalian cells while rarely extended to plant engineering. Cationic polymers and lipids have been widely utilized to efficiently deliver DNA and siRNA into mammalian cells. However, their application in plant cells is limited due to the different membrane structures and the presence of plant cell walls. In this study, we developed the cationic, α-helical polypeptide that can effectively deliver DNA into both isolated Arabidopsis thaliana protoplasts and intact leaves. The PPABLG was able to condense DNA to form nanocomplexes, and they exhibited significantly improved transfection efficiencies compared with commercial transfection reagent Lipofectamine 2000 and classical cell penetrating peptides such as poly(L-lysine), HIV-TAT, arginine9, and poly(L-arginine). This study therefore widens the utilities of helical polypeptide as a unique category of gene delivery materials, and may find their promising applications toward plant gene delivery.


α-helical polypeptide plant gene delivery protoplast intact leaves transfection 


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L. Y. acknowledges the support from the National Natural Science Foundation of China (Grant Nos.51403145 and 51573123), the Science and Technology Department of Jiangsu Province (BK20140333), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). J. C. acknowledges support from the NSF (CHE-1153122), the NIH (NIH Director’s New Innovator Award 1DP2OD007246 and 1R21EB013379). J. C. also acknowledges support from Dr. Ray Zielinski (Department of Plant Biology, University of Illinois at Urbana–Champaign) for the provision of plants and plasmid DNA, as well as the technic of protoplasts isolation.


  1. 1.
    Borchert R, Renner S S, Calle Z, Navarrete D, Tye A, Gautier L, Spichiger R, von Hildebrand P. Photoperiodic induction of synchronous flowering near the Equator. Nature, 2005, 433(7026): 627–629CrossRefGoogle Scholar
  2. 2.
    Dubreuil G, Magliano M, Dubrana M P, Lozano J, Lecomte P, Favery B, Abad P, Rosso M N. Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode. Journal of Experimental Botany, 2009, 60(14): 4041–4050CrossRefGoogle Scholar
  3. 3.
    Pasupathy K, Lin S, Hu Q, Luo H, Ke P C. Direct plant gene delivery with a poly(amidoamine) dendrimer. Biotechnology Journal, 2008, 3(8): 1078–1082CrossRefGoogle Scholar
  4. 4.
    Hussain M M, Melcher U, Essenberg R C. Infection of evacuolated turnip protoplasts with liposome-packaged cauliflower mosaicvirus. Plant Cell Reports, 1985, 4(2): 58–62CrossRefGoogle Scholar
  5. 5.
    Li Y, Cui H, Song Y, Li Y, Huang J. Transient expression of exogenous gene into plant cell mediated by PEI nanovector. Agricultural Sciences in China, 2011, 10(6): 820–826CrossRefGoogle Scholar
  6. 6.
    Boynton J E, Gillham N W, Harris E H, Hosler J P, Johnson A M, Jones A R, Randolphanderson B L, Robertson D, Klein T M, Shark K B, Sanford J C. Chloroplast transformation in chlamydomonas with high-velocity microprojectiles. Science, 1988, 240(4858): 1534–1538CrossRefGoogle Scholar
  7. 7.
    Carqueijeiro I, Masini E, Foureau E, Sepulveda L J, Marais E, Lanoue A, Besseau S, Papon N, Clastre M, de Bernonville T D, Glevarec G, Atehortua L, Oudin A, Courdavault V. Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors. Plant Biology, 2015, 17(6): 1242–1246CrossRefGoogle Scholar
  8. 8.
    Koop H U, Steinmuller K, Wagner H, Rossler C, Eibl C, Sacher L. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta, 1996, 199(2): 193–201CrossRefGoogle Scholar
  9. 9.
    Wang F, Liu J, Tong C, Wang Q, Tang D, Yi L, Wang L L, Liu XM. Magnetic nanoparticle as rice transgene vector mediated by electroporation. Chinese Journal of Analytical Chemistry, 2010, 38(5): 617–621Google Scholar
  10. 10.
    Miranda A, Janssen G, Hodges L, Peralta E G, Ream W. Agrobacterium-tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism. Journal of Bacteriology, 1992, 174(7): 2288–2297CrossRefGoogle Scholar
  11. 11.
    Rakoczy-Trojanowska M. Alternative methods of plant transformation. Cellular & Molecular Biology Letters, 2002, 7(3): 849–858Google Scholar
  12. 12.
    Nair R, Varghese S H, Nair B G, Maekawa T, Yoshida Y, Kumar D S. Nanoparticulate material delivery to plants. Plant Science, 2010, 179(3): 154–163CrossRefGoogle Scholar
  13. 13.
    Chugh A, Eudes F. Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos. FEBS Journal, 2008, 275(10): 2403–2414CrossRefGoogle Scholar
  14. 14.
    Chen C, Chou J, Liu B, Chang M, Lee H. Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Letters, 2007, 581(9): 1891–1897CrossRefGoogle Scholar
  15. 15.
    Lakshmanan M, Kodama Y, Yoshizumi T, Sudesh K, Numata K. Rapid and efficient gene delivery into plant cells using designed peptide carriers. Biomacromolecules, 2013, 14(1): 10–16CrossRefGoogle Scholar
  16. 16.
    Hariton-Gazal E, Rosenbluh J, Graessmann A, Gilon C, Loyter A. Direct translocation of histone molecules across cell membranes. Journal of Cell Science, 2003, 116(22): 4577–4586CrossRefGoogle Scholar
  17. 17.
    Rosenbluh J, Singh S K, Gafni Y, Graessmann A, Loyter A. Nonendocytic penetration of core histones into petunia protoplasts and cultured cells: A novel mechanism for the introduction of macromolecules into plant cells. Biochimica et Biophysica Acta-Biomembranes, 2004, 1664(2): 230–240CrossRefGoogle Scholar
  18. 18.
    Wei Y, Niu J, Huan L, Huang A, He L, Wang G. Cell penetrating peptide can transport dsRNA into microalgae with thin cell walls. Algal Research-Biomass Biofuels and Bioproducts, 2015, 8: 135–139Google Scholar
  19. 19.
    Hyman J M, Geihe E I, Trantow B M, Parvin B, Wender P A. A molecular method for the delivery of small molecules and proteins across the cell wall of algae using molecular transporters. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(33): 13225–13230CrossRefGoogle Scholar
  20. 20.
    Fonseca S B, Pereira MP, Kelley S O. Recent advances in the use of cell-penetrating peptides for medical and biological applications. Advanced Drug Delivery Reviews, 2009, 61(11): 953–964CrossRefGoogle Scholar
  21. 21.
    Elsner MB, Herold HM, Muller-Herrmann S, Bargel H, Scheibel T. Enhanced cellular uptake of engineered spider silk particles. Biomaterials Science, 2015, 3(3): 543–551CrossRefGoogle Scholar
  22. 22.
    Saw P E, Ko Y T, Jon S. Efficient liposomal nanocarrier-mediated oligodeoxynucleotide delivery involving dual use of a cellpenetrating peptide as a packaging and intracellular delivery agent. Macromolecular Rapid Communications, 2010, 31(13): 1155–1162CrossRefGoogle Scholar
  23. 23.
    Patra S, Roy E, Madhuri R, Sharma P K. The next generation cellpenetrating peptide and carbon dot conjugated nano-liposome for transdermal delivery of curcumin. Biomaterials Science, 2016, 4(3): 418–429CrossRefGoogle Scholar
  24. 24.
    Chen S, Rong L, Jia H Z, Qin S Y, Zeng X, Zhuo R X, Zhang X Z. Co-delivery of proapoptotic peptide and p53 DNA by reductionsensitive polypeptides for cancer therapy. Biomaterials Science, 2015, 3(5): 753–763CrossRefGoogle Scholar
  25. 25.
    Gabrielson N P, Lu H, Yin L, Li D, Wang F, Cheng J. Reactive and bioactive cationic α-helical polypeptide template for nonviral gene delivery. Angewandte Chemie International Edition, 2012, 51(5): 1143–1147CrossRefGoogle Scholar
  26. 26.
    Lu H, Wang J, Bai Y, Lang J W, Liu S, Lin Y, Cheng J. Ionic polypeptides with unusual helical stability. Nature Communications, 2011, 2: 206CrossRefGoogle Scholar
  27. 27.
    Zheng N, Song Z, Liu Y, Zhang R, Zhang R, Yao C, Uckun F M, Yin L, Cheng J. Redox-responsive, reversibly-crosslinked thiolated cationic helical polypeptides for efficient siRNA encapsulation and delivery. Journal of Controlled Release, 2015, 205: 231–239CrossRefGoogle Scholar
  28. 28.
    Zheng N, Yin L, Song Z, Ma L, Tang H, Gabrielson N P, Lu H, Cheng J. Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting. Biomaterials, 2014, 35(4): 1302–1314CrossRefGoogle Scholar
  29. 29.
    Yin L, Tang H, Kim K H, Zheng N, Song Z, Gabrielson N P, Lu H, Cheng J. Light-responsive helical polypeptides capable of reducing toxicity and unpacking DNA: Toward nonviral gene delivery. Angewandte Chemie International Edition, 2013, 52(35): 9182–9186CrossRefGoogle Scholar
  30. 30.
    Yin L, Song Z, Kim K H, Zheng N, Gabrielson N P, Cheng J. Nonviral gene delivery via membrane-penetrating, mannose-targeting supramolecular self-assembled nanocomplexes. Advanced Materials, 2013, 25(22): 3063–3070CrossRefGoogle Scholar
  31. 31.
    Rondeau-Mouro C, Defer D, Leboeuf E, Lahaye M. Assessment of cell wall porosity in Arabidopsis thaliana by NMR spectroscopy. International Journal of Biological Macromolecules, 2008, 42(2): 83–92CrossRefGoogle Scholar
  32. 32.
    Gunl M, Pauly M. AXY3 encodes a alpha-xylosidase that impacts the structure and accessibility of the hemicellulose xyloglucan in Arabidopsis plant cell walls. Planta, 2011, 233(4): 707–719CrossRefGoogle Scholar
  33. 33.
    Lu S, Hu J, Liu B, Lee C, Li J, Chou J, Lee H J. Arginine-rich intracellular delivery peptides synchronously deliver covalently and noncovalently linked proteins into plant cells. Journal of Agricultural and Food Chemistry, 2010, 58(4): 2288–2294CrossRefGoogle Scholar
  34. 34.
    Eudes F, Chugh A. Cell-penetrating peptides: From mammalian to plant cells. Plant Signaling & Behavior, 2008, 3(8): 549–550CrossRefGoogle Scholar
  35. 35.
    Battey N H, James N C, Greenland A J, Brownlee C. Exocytosis and endocytosis. Plant Cell, 1999, 11(4): 643–660CrossRefGoogle Scholar
  36. 36.
    Chiu W L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J. Engineered GFP as a vital reporter in plants. Current Biology, 1996, 6(3): 325–330CrossRefGoogle Scholar
  37. 37.
    Pedelacq J D, Cabantous S, Tran T, Terwilliger T C, Waldo G S. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology, 2006, 24(1): 79–88CrossRefGoogle Scholar
  38. 38.
    Liu S, Yang J X, Ren H Q, O’Keeffe-Ahern J, Zhou D Z, Zhou H, Chen J T, Guo T Y. Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(Llysine). Biomaterials Science, 2016, 4(3): 522–532CrossRefGoogle Scholar
  39. 39.
    Mintzer M A, Simanek E E. Nonviral vectors for gene delivery. Chemical Reviews, 2009, 109(2): 259–302CrossRefGoogle Scholar
  40. 40.
    Navarro E, Baun A, Behra R, Hartmann N B, Filser J, Miao A J, Quigg A, Santschi P H, Sigg L. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology (London, England), 2008, 17(5): 372–386CrossRefGoogle Scholar
  41. 41.
    Fleischer A, O’Neill M A, Ehwald R. The pore size of nongraminaceous plant cell walls is rapidly decreased by borate ester cross-linking of the pectic polysaccharide rhamnogalacturonan II. Plant Physiology, 1999, 121(3): 829–838CrossRefGoogle Scholar
  42. 42.
    Tang H, Yin L, Kim K H, Cheng J. Helical poly(arginine) mimics with superior cell-penetrating and molecular transporting properties. Chemical Science (Cambridge), 2013, 4(10): 3839–3844CrossRefGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Materials Science and EngineeringUniversity of Illinois at Urbana-ChampaignChampaignUSA
  2. 2.State Key Laboratory of Fine Chemicals, Department of Polymer Science and Engineering, School of Chemical EngineeringDalian University of TechnologyDalianChina
  3. 3.Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Collaborative Innovation Center of Suzhou Nano Science and TechnologySoochow UniversitySuzhouChina

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