A nano-biomimetic transformation system enables in planta expression of a reporter gene in mature plants and seeds

Abstract

Plant genetic engineering will be essential to decipher the genomic basis of complex traits, optimize crop genomics, and enable plant-based production of recombinant proteins. However, established plant transformation approaches for bioengineering are fraught with limitations. Although nanoparticle-mediated methods show great promise for advancing plant biotechnology, many engineered nanomaterials can have cytotoxic and ecological effects. Here, we demonstrate the efficient uptake of a nano-biomimetic carrier of plasmid DNA and transient expression of a reporter gene in leaves of Arabidopsis, common ice plant and tobacco, as well as in the developing seed tissues of Arabidopsis, field mustard, barley, and wheat. The nano-biomimetic transformation system described here has all the advantages of other nanoparticle-mediated approaches for passive delivery of genetic cargo into a variety of plant species and is also nontoxic to cells and to the environment for diverse biotechnological applications in plant biology and crop science.

Fig. 1. Morphologies of synthesized nanohydroxyapatites (nHAs). (a) Scanning Electron Microscope (SEM) image of nHAs. (b) Transmission Electron Microscope (TEM) image of nHAs. (c) TEM image of arginine-functionalized nHAs (R-nHA). Scale bars: 100 nm (a); and 20 nm (b and c). (d) Distribution of particle diameters (nm) in nHAs (red colored columns) and R-nHAs (black colored columns). (e) Distribution of particle lengths (nm) in nHAs (red colored columns) and R-nHAs (black colored columns).

Fig. 2. Physicochemical characteristics of nanohydroxyapatites (nHAs). (a) Powdered X-ray diffraction (PXRD) pattern of nHAs and R-nHAs (functionalized with arginine). (b) Energy Dispersive Spectroscopy (EDS) spectra of nHAs, where measured Ca/P ratio is ∼1.71.

Fig. 3. Conjugation of DNA plasmid (pDNA) with nanohydroxyapatites (nHAs). (a) Conjugation efficiency assay of pDNA and R-nHAs by measuring the optical density of the supernatant (i.e., unbound pDNA in an aqueous solution of water) at 260 nm indicates the 1 : 200 mass ratio conjugates have the highest conjugating efficiencies. Error bars are standard error of the mean (n = 3). (b) Agarose gel electrophoretogram of pDNA–R-nHA conjugates confirms the 1 : 200 mass ratio conjugates is the most efficient ratio as indicated by the bright intensity of the band in lane 6. Lane 1 – 1 : 10 mass ratio. Lane 2 – 1 : 30 mass ratio. Lane 3 – 1 : 50 mass ratio. Lane 4 – 1 : 70 mass ratio. Lane 5 – 1 : 100 mass ratio. Lane 6 – 1 : 200 mass ratio. Lane 7 – pDNA. Faint bands indicate relaxed and supercoiled (bright bands) conformations of pDNA or pDNA–R-nHA conjugates. (c) DNase assay of conjugates suggests nHAs might reduce or inhibit endonuclease activity, especially relaxed forms of the plasmid. Lane 1 – pDNA–R-nHA conjugates with DNase treatment. Lane 2 – pDNA–R-nHA conjugates without DNase treatment. Lane 3 – pDNA–nHA conjugates with DNase treatment. Lane 4 – pDNA–nHA conjugates without DNase treatment. Lane 5 – pDNA with DNase treatment. Lane 6 – pDNA without DNase treatment. Faint bands indicate relaxed and supercoiled (bright bands) conformations of pDNA or pDNA–nHA conjugates.

Fig. 4. Plasmid DNA (pDNA) delivery into mature leaves with arginine-functionalized nanohydroxyapatites (R-nHAs) and subsequent GUS expression. (a) Model of uptake and translocation of DNA–nHA conjugates and subsequent expression of introduced gene(s) in a plant cell. Dotted-line arrows represent trafficking of complexes, whereas solid-line arrows represent expression of introduced gene(s). DNA–nHA – plasmids or PCR amplicons conjugated to nHA. ML – middle lamella. PCW – primary cell wall. SCW – secondary cell wall. PM – plasma membrane. nDNA – nuclear DNA. mRNA – messenger RNA. GUS – stained GUS protein product. GFP – GFP product. (b–h) Transient GUS expression in leaves of mature tobacco (Nicotiana benthamiana) and Arabidopsis thaliana after in vivo infiltration of pDNA–R-nHA conjugates of healthy plants using a syringe, and Arabidopsis, and ice plant (Mesembryanthemum crystallinum) after vacuum infiltration of pDNA–R-nHA conjugates in detached leaves from healthy plants: (b) control (untreated) – tobacco; (c) Agrobacterium-mediated expression of GUS in tobacco; (d) pDNA–R-nHA-mediated expression of GUS in tobacco; (e) control (untreated – infiltrated with water) – Arabidopsis; (f) pDNA–R-nHA-mediated expression of GUS in Arabidopsis; (g) control (untreated – infiltrated with water) – ice plant; and (h) pDNA–R-nHA-mediated expression of GUS in ice plant. Blue spots or patches (arrows) represent histochemical staining of GUS.

Fig. 5. Plasmid DNA (pDNA) delivery into seeds with arginine-functionalized nanohydroxyapatites (R-nHAs) via incubation in a solution of pDNA–R-nHA conjugates and subsequent GUS expression. (a) Control (untreated – incubated in water) – barley (Hordeum vulgare) seed. (b) Transient GUS expression in imbibed barley seed after incubation in a solution of pDNA–R-nHA conjugates. (c) Magnification at 25× of the barley seedling. (d) Control (untreated – incubated in water) – wheat (Triticum aestivum) seed. (e) Transient GUS expression in imbibed wheat seed after incubation in a solution of pDNA–R-nHA conjugates. Arrow indicates embryonic tissue expressing GUS. (f) Control (untreated – incubated in water) – Brassica rapa seed at 8× magnification. (g) Transient GUS expression in imbibed Brassica rapa seed after incubation in a solution of pDNA–R-nHA conjugates. Magnification at 8× of developing seedling. (h) Magnification at 15× of the seedling radical. Blue spots or patches (arrows) represent histochemical staining of GUS in all the panels.

Fig. 6. Delivery of plasmid DNA (pDNA) with green fluorescent protein gene into root cells of seedlings with arginine-functionalized nanohydroxyapatites (R-nHAs) via incubation in a solution of pDNA–R-nHA conjugates and subsequent GFP expression. (a) Root cells of 4 d old Arabidopsis seedling after incubation in water (i.e., control). (b) Transient GFP expression in imbibed root cells of 4 d Arabidopsis seedling after incubation in a solution of pDNA–R-nHA conjugates. (c) Root cells of 4 d field mustard (Brassica rapa) seedling after incubation in water (i.e., control). (d) Transient GFP expression in imbibed root cells of 4 d field mustard seedling after incubation in a solution of pDNA–R-nHA conjugates. Green patches (arrows) indicate GFP protein expression in cytoplasm of root cells in panels b and d, whereas the green outline of the cell is due to autofluorescence of lignin in the cell wall in all panels. All images are at 100× magnification. Scale bars: 20 μm.