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Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

Postal Subscription Code 80-969

2018 Impact Factor: 2.809

Front. Chem. Sci. Eng.    2017, Vol. 11 Issue (4) : 521-528    https://doi.org/10.1007/s11705-017-1612-8
RESEARCH ARTICLE
Gene delivery into isolated Arabidopsis thaliana protoplasts and intact leaves using cationic, α-helical polypeptide
Nan Zheng1,2, Ziyuan Song1, Yang Liu1, Lichen Yin3(), Jianjun Cheng1()
1. Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, IL 61801, USA
2. State Key Laboratory of Fine Chemicals, Department of Polymer Science and Engineering, School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China
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 Technology, Soochow University, Suzhou 215123, China
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Abstract

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.

Keywords α-helical polypeptide      plant gene delivery      protoplast      intact leaves      transfection     
Corresponding Author(s): Lichen Yin,Jianjun Cheng   
Online First Date: 13 January 2017    Issue Date: 06 November 2017
 Cite this article:   
Nan Zheng,Ziyuan Song,Yang Liu, et al. Gene delivery into isolated Arabidopsis thaliana protoplasts and intact leaves using cationic, α-helical polypeptide[J]. Front. Chem. Sci. Eng., 2017, 11(4): 521-528.
 URL:  
https://academic.hep.com.cn/fcse/EN/10.1007/s11705-017-1612-8
https://academic.hep.com.cn/fcse/EN/Y2017/V11/I4/521
Fig.1  scheme 1(A) Synthetic routes of PPABLG; (B) Schematic illustration of the formation of PPABLG/DNA complexes and gene delivery in both isolated protoplasts and intact leaves
Fig.2  (A) CD spectrum of PPABLG in deionized water (0.2 mg/mL) at pH 7.0; (B) DNA condensation by PPABLG at different N/P ratios as evaluated by the gel retardation assay. N represents naked DNA; (C) EB exclusion assay showing the condensation of DNA in the PPABLG/DNA complexes; (D) particle size and zeta potential of PPABLG/DNA complexes at various N/P ratios; (E) SEM image of PPABLG/DNA complexes at the N/P ratio of 30 (bar= 200 nm); (F) stability of PPABLG/DNA complexes (N/P= 30) following various incubation time at 37 °C
Fig.3  (A) Transfection efficiencies of PPABLG/DNA complexes in Arabidopsis thaliana protoplast cells at various DNA amount and PPABLG/DNA N/P ratios (n = 3); (B) transfection efficiencies of various complexes in Arabidopsis thaliana protoplast cells (n = 3). DNA amount was maintained constant at 2 µg/105 cells. N represents naked DNA. LPF, PLL, TAT, Arg9, and PLR at the N/P ratios of 5, 8, 15, 15 and 10 served as controls, respectively
Fig.4  Transfection efficiencies of polypeptide/DNA complexes in intact leaves as observed by CLSM. Spongy mesophyll cells ofArabidopsis thaliana leaves infiltrated with (A) ultrapure water; (B) naked DNA; (C) PPABLG/DNA complexes (N/P= 20); (D) PPABLG/DNA complexes (N/P= 30); (E) PPABLG/DNA complexes (N/P= 40); (F) LPF/DNA complexes (N/P= 5); (G) PLL/DNA complexes (N/P= 8); (H) TAT/DNA complexes (N/P= 15); (I) Arg9/DNA complexes (N/P= 15); (J) PLR/DNA complexes (N/P= 10). C, D, and E showed GFP expression (green, white arrows) in the cytosol, which was notably different from the autofluorescence of chloroplasts (red)
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): 627–629
https://doi.org/10.1038/nature03259
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): 4041–4050
https://doi.org/10.1093/jxb/erp237
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): 1078–1082
https://doi.org/10.1002/biot.200800021
4 Hussain M M, Melcher  U, Essenberg R C . Infection of evacuolated turnip protoplasts with liposome-packaged cauliflower mosaic-virus. Plant Cell Reports, 1985, 4(2): 58–62
https://doi.org/10.1007/BF00269206
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): 820–826
https://doi.org/10.1016/S1671-2927(11)60067-9
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): 1534–1538
https://doi.org/10.1126/science.2897716
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): 1242–1246
https://doi.org/10.1111/plb.12380
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): 193–201
https://doi.org/10.1007/BF00196559
9 Wang F, Liu  J, Tong C ,  Wang Q, Tang  D, Yi L ,  Wang L L ,  Liu X M . Magnetic nanoparticle as rice transgene vector mediated by electroporation. Chinese Journal of Analytical Chemistry, 2010, 38(5): 617–621
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): 2288–2297
https://doi.org/10.1128/jb.174.7.2288-2297.1992
11 Rakoczy-Trojanowska M . Alternative methods of plant transformation. Cellular & Molecular Biology Letters, 2002, 7(3): 849–858
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): 154–163
https://doi.org/10.1016/j.plantsci.2010.04.012
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): 2403–2414
https://doi.org/10.1111/j.1742-4658.2008.06384.x
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): 1891–1897
https://doi.org/10.1016/j.febslet.2007.03.076
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): 10–16
https://doi.org/10.1021/bm301275g
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): 4577–4586
https://doi.org/10.1242/jcs.00757
17 Rosenbluh J, Singh  S K, Gafni  Y, Graessmann A ,  Loyter A . Non-endocytic 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): 230–240
https://doi.org/10.1016/j.bbamem.2004.06.003
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: 135–139
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): 13225–13230
https://doi.org/10.1073/pnas.1202509109
20 Fonseca S B, Pereira  M P, Kelley  S O. Recent advances in the use of cell-penetrating peptides for medical and biological applications. Advanced Drug Delivery Reviews, 2009, 61(11): 953–964
https://doi.org/10.1016/j.addr.2009.06.001
21 Elsner M B, Herold  H M, Muller-Herrmann  S, Bargel H ,  Scheibel T . Enhanced cellular uptake of engineered spider silk particles. Biomaterials Science, 2015, 3(3): 543–551
https://doi.org/10.1039/C4BM00401A
22 Saw P E, Ko  Y T, Jon  S. Efficient liposomal nanocarrier-mediated oligodeoxynucleotide delivery involving dual use of a cell-penetrating peptide as a packaging and intracellular delivery agent. Macromolecular Rapid Communications, 2010, 31(13): 1155–1162
https://doi.org/10.1002/marc.200900861
23 Patra S, Roy  E, Madhuri R ,  Sharma P K . The next generation cell-penetrating peptide and carbon dot conjugated nano-liposome for transdermal delivery of curcumin. Biomaterials Science, 2016, 4(3): 418–429
https://doi.org/10.1039/C5BM00433K
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 reduction-sensitive polypeptides for cancer therapy. Biomaterials Science, 2015, 3(5): 753–763
https://doi.org/10.1039/C5BM00046G
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): 1143–1147
https://doi.org/10.1002/anie.201104262
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: 206
https://doi.org/10.1038/ncomms1209
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: 231–239
https://doi.org/10.1016/j.jconrel.2015.02.014
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): 1302–1314
https://doi.org/10.1016/j.biomaterials.2013.09.090
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): 9182–9186
https://doi.org/10.1002/anie.201302820
30 Yin L, Song  Z, Kim K H ,  Zheng N ,  Gabrielson N P ,  Cheng J . Non-viral gene delivery via membrane-penetrating, mannose-targeting supramolecular self-assembled nanocomplexes. Advanced Materials, 2013, 25(22): 3063–3070
https://doi.org/10.1002/adma.201205088
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): 83–92
https://doi.org/10.1016/j.ijbiomac.2007.09.020
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): 707–719
https://doi.org/10.1007/s00425-010-1330-7
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): 2288–2294
https://doi.org/10.1021/jf903039j
34 Eudes F, Chugh  A. Cell-penetrating peptides: From mammalian to plant cells. Plant Signaling & Behavior, 2008, 3(8): 549–550
https://doi.org/10.4161/psb.3.8.5696
35 Battey N H, James  N C, Greenland  A J, Brownlee  C. Exocytosis and endocytosis. Plant Cell, 1999, 11(4): 643–660
https://doi.org/10.1105/tpc.11.4.643
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): 325–330
https://doi.org/10.1016/S0960-9822(02)00483-9
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): 79–88
https://doi.org/10.1038/nbt1172
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(L-lysine). Biomaterials Science, 2016, 4(3): 522–532
https://doi.org/10.1039/C5BM00530B
39 Mintzer M A, Simanek  E E. Nonviral vectors for gene delivery. Chemical Reviews, 2009, 109(2): 259–302
https://doi.org/10.1021/cr800409e
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): 372–386
https://doi.org/10.1007/s10646-008-0214-0
41 Fleischer A, O’Neill  M A, Ehwald  R. The pore size of non-graminaceous plant cell walls is rapidly decreased by borate ester cross-linking of the pectic polysaccharide rhamnogalacturonan II. Plant Physiology, 1999, 121(3): 829–838
https://doi.org/10.1104/pp.121.3.829
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): 3839–3844
https://doi.org/10.1039/c3sc51328a
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