Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle-Complex-Mediated DNA Delivery to Specific Plant Organelles

Abstract

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants.

Keywords: Gene Technology; Micelles; Nanotechnology; Peptides; Zwitterions.

Figure 1

Schematic overview of pDNA delivery to specific organelles in plants through the combination of a zwitterionic liquid and peptide‐displaying micelle complexes. Zwitterionic liquid pretreatment is intended to enhance cell wall permeability, allowing the efficient translocation of peptide‐displaying micelle complexes across the cell wall into intracellular target organelles.

Figure 2

Biocompatibility and cellulose‐dissolving ability of ZIL. A) Chemical structures of ZIL, IL‐1, and IL‐2. B) Viability of A. thaliana seedlings pretreated for 3 h with various concentrations of ZIL, IL‐1, or IL‐2, followed by 24 h of incubation. The viability was determined by the Evans blue assay. Data from four biological replicates are represented as the mean±standard error values. Statistical significance: * P<0.05, ** P<0.01 based on Dunnett's T3 test (n=4). C) 1D WAXD profiles obtained from cellulose microcrystals 2 h after pretreatment with 0, 200, or 400 mM ZIL. D) AFM height images of cellulose microcrystals pretreated with 0, 200, or 400 mM ZIL for 2 h on a highly oriented pyrolytic graphite (HOPG) substrate. Scale bars, 500 nm. Color bars represent the height of the cellulose microcrystal.

Figure 3

Effect of ZIL on cell wall permeability in plants. A) CLSM images of cellulose fibrils stained with calcofluor white in epidermal cells of A. thaliana cotyledons after pretreatment with 0, 200, or 400 mM ZIL for 3 h. Scale bars, 5 μm. B) CLSM images showing FM4‐64 fluorescence quenching by TB in epidermal cells of A. thaliana cotyledons pretreated for 3 h with 0, 200, or 400 mM ZIL. Scale bars, 20 μm. C) Relative fluorescence intensity of FM4‐64 at several TB concentrations shown in B. Data from four biological replicates are represented as the mean±standard error values. D) Stern–Volmer plots of FM4‐64 fluorescence quenching by TB in epidermal cells of A. thaliana cotyledons pretreated for 3 h with 0, 200, or 400 mM ZIL. The slope of the regression line indicates the quenching efficiency. Data from four biological replicates are represented as the mean ± standard error values.

Figure 4

Effects of ZIL on the nuclear transfection efficiency of CPP‐MC in plants. A) Schematic illustration of CPP‐MC‐mediated reporter gene (GFP or NanoLuc^TM luciferase (Nluc)) transfection of the nucleus in ZIL‐pretreated plants. B) CLSM images showing GFP expression in epidermal cells in ZIL‐pretreated A. thaliana cotyledons 24 h after transfection with CPP‐MC or naked pDNA. C, control sample transfected with the naked pDNA. Scale bars, 40 μm. Chlorophyll fluorescence and bright‐field images corresponding to the GFP fluorescent images are shown in Figure S7. C) Boxplot representation of the relative transfection efficiency of CPP‐MC based on the Nluc expression levels in ZIL‐untreated and ZIL‐pretreated A. thaliana seedlings 24 h post‐infiltration: the boxes represent the interquartile range, the lines within the boxes represent the median values, and the upper and lower whiskers represent the highest and lowest values, respectively. Statistical significance compared to the control (ZIL conc., 0 mM): ** P<0.01, **** P<0.0001 based on Dunnett's T3 test (n=30 biological replicates). D) Intensity size distributions, Z‐average diameters, and PDI of CPP‐MC‐S and CPP‐MC‐L based on DLS measurements (n=3). E) AFM height images of CPP‐MC‐S and CPP‐MC‐L. Scale bars, 200 nm. Color bars represent the height of the micelle. F) Boxplot representation of the relative transfection efficiency of CPP‐MC‐S and CPP‐MC‐L based on the Nluc expression levels in ZIL‐untreated and ZIL‐pretreated A. thaliana seedlings 24 h post‐infiltration. Statistical significance between the ZIL‐untreated and ZIL‐pretreated samples for each micelle was determined by Dunnett's T3 test (n=30 biological replicates).

Figure 5

Effects of ZIL on the chloroplast transfection efficiency of CTP/CPP‐MC in plants. A) Schematic illustration of CTP/CPP‐MC‐mediated reporter gene (GFP or Renilla luciferase (Rluc)) transfection of chloroplasts in ZIL‐pretreated plants. B) CLSM images showing GFP expression in epidermal cells in ZIL‐untreated and ZIL‐pretreated A. thaliana cotyledons 24 h after transfection with CTP/CPP‐MC or controls (naked pDNA or CTP/CPP‐MC containing pDNA for nucleus transfection (P35S‐GFP‐Tnos)). Scale bars, 40 μm. C) Boxplot representation of the relative transfection efficiency of each system based on the Rluc expression levels in ZIL‐pretreated A. thaliana seedlings 24 h post‐infiltration. Statistical significance compared to the control (CTP/CPP‐MC, ZIL (−)): **** P<0.0001 based on Dunnett's T3 test (n=20 biological replicates).