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. 2022 May 7;20(1):219.
doi: 10.1186/s12951-022-01443-4.

Construction and application of star polycation nanocarrier-based microRNA delivery system in Arabidopsis and maize

Jia Yang #  1 , Shuo Yan #  2 , Shipeng Xie  1 , Meizhen Yin  3 , Jie Shen  2 , Zhaohu Li  1 , Yuyi Zhou  4 , Liusheng Duan  5   6
Affiliations

Construction and application of star polycation nanocarrier-based microRNA delivery system in Arabidopsis and maize

Jia Yang et al. J Nanobiotechnology. .

Abstract

Background: MicroRNA (miRNA) plays vital roles in the regulation of both plant architecture and stress resistance through cleavage or translation inhibition of the target messenger RNAs (mRNAs). However, miRNA-induced gene silencing remains a major challenge in vivo due to the low delivery efficiency and instability of miRNA, thus an efficient and simple method is urgently needed for miRNA transformation. Previous researches have constructed a star polycation (SPc)-mediated transdermal double-stranded RNA (dsRNA) delivery system, achieving efficient dsRNA delivery and gene silencing in insect pests.

Results: Here, we tested SPc-based platform for direct delivery of double-stranded precursor miRNA (ds-MIRNA) into protoplasts and plants. The results showed that SPc could assemble with ds-MIRNA through electrostatic interaction to form nano-sized ds-MIRNA/SPc complex. The complex could penetrate the root cortex and be systematically transported through the vascular tissue in seedlings of Arabidopsis and maize. Meanwhile, the complex could up-regulate the expression of endocytosis-related genes in both protoplasts and plants to promote the cellular uptake. Furthermore, the SPc-delivered ds-MIRNA could efficiently increase mature miRNA amount to suppress the target gene expression, and the similar phenotypes of Arabidopsis and maize were observed compared to the transgenic plants overexpressing miRNA.

Conclusion: To our knowledge, we report the first construction and application of star polycation nanocarrier-based platform for miRNA delivery in plants, which explores a new enable approach of plant biotechnology with efficient transformation for agricultural application.

Keywords: Gene silencing; MiRNA delivery; Nanoparticle; Plant biotechnology; Star polycation.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1
Synthesis route of Pt-Br and SPc
Fig. 1
Fig. 1
Self-assembly and characterization of ds-MIRNA/SPc complex. a Gel electrophoresis assay of ds-MIR164b retardation by SPc. The ds-MIR164b/SPc complexes (ds-MIRNA: 3 μg) were prepared at the mass ratios of 5:1, 3:1, 1:1 and 1:3 (ds-MIRNA: SPc), respectively. b Size distribution measurement of ds-MIR164b/SPc complex at the mass ratio of 1:1. c Zeta potential of ds-MIR164b/SPc complex at the mass ratio of 1:1. The “***” indicates the significant difference according to independent t-test at P < 0.001. d TEM images of ds-MIR164b/SPc complex at the mass ratio of 1:1. Small complexes were enlarged
Fig. 2
Fig. 2
Enhanced delivery of SPc-loaded ds-MIRNA into Arabidopsis roots. a Schematic diagram of root application and representative confocal images of Arabidopsis roots at 6 after the treatment of various formulations. Red arrow and box indicate the application and observation site, respectively. Red: ds-MIRNA; Green: GFP; Scale bar: 20 µm. b Representative confocal images of Arabidopsis roots at 12 h after the treatment of various formulations. Red: ds-MIRNA; Scale bar: 50 µm. c Fluorescent quantitative analysis of Arabidopsis roots in a. Different letters on columns indicate significant differences (Duncan’s multiple range test, P < 0.05)
Fig. 3
Fig. 3
Enhanced delivery of SPc-loaded ds-MIRNA into maize roots. a Schematic diagram of root application. Red arrow and box indicate the application and observation site, respectively. b Representative longitudinal section images of roots and fluorescent analysis of SPc-delivered ds-MIRNA at 12 h after the treatment. Right and left scale bars indicate 200 and 20 µm, respectively. Different letters on columns indicate significant differences (Duncan’s multiple range test, P < 0.05). c Representative cross section images of roots and fluorescent analysis of SPc-delivered ds-MIRNA at 12 h after the treatment. Right and left scale bars indicate 200 and 100 µm, respectively
Fig. 4
Fig. 4
The SPc-delivered ds-MIRNA internalizes into protoplasts and maize through endocytosis. a Differentially expressed genes related to endocytosis in protoplasts. The treated protoplasts were collected after 2 h incubation. The housekeeping gene ZmUBC was used as the reference gene. Triplicate biological replicates were used for each treatment. Different letters on columns indicate significant differences among the treatments of control, ds-MIRNA and ds-MIRNA/SPc complex (Duncan’s multiple range test, P < 0.05). b Differentially expressed genes related to endocytosis in maize. The ds-MIR164b/SPc complex was applied to the root surface of maize every 24 h, and the expression of above endocytosis-related genes was determined on 4 days after the treatment. c Schematic illustration of the ds-MIRNA/SPc complex delivery into cells. The negatively-charged ds-MIRNA can be assembled with positively-charged SPc into ds-MIRNA/SPc complex, which is delivered into cells through endocytosis. The endocytosis-related genes were up-regulated, and the location of encoded proteins is shown
Fig. 5
Fig. 5
Ds-MIRNA/SPc complex-mediated gene silencing in Arabidopsis. a QRT-PCR assay for MIR166a and miR166a. The ds-MIR166a/SPc complex was applied to the root surface of Arabidopsis every 24 h, and the samples were collected on 6 days after the treatment. The housekeeping gene AtActin-2 was used as the reference gene. Triplicate biological replicates were used for each treatment. Different letters on columns indicate significant differences (Duncan’s multiple range test, P < 0.05). b QRT-PCR assay for target genes. c Photos of Arabidopsis seedlings among various treatments. Scale bar: 500 μm. d Development evaluation of SAM by measuring the leaf # 5 and 6. e The photos of Col (wild type) and transgenic Arabidopsis strain overexpressing Ath-miR166a, which were taken with 10-day-old seedlings. Scale bar: 1 mm
Fig. 6
Fig. 6
Ds-MIRNA/SPc complex-mediated gene silencing in maize. a QRT-PCR assay for MIR164b and miR164b. The ds-MIR164b/SPc complex was applied to the root surface of maize every 24 h, and the samples were collected on 4 days after the treatment. Triplicate biological replicates were used for each treatment. The housekeeping gene ZmUBC was used as the reference gene. Different letters on columns indicate significant differences (Duncan’s multiple range test, P < 0.05). b QRT-PCR assay for target gene NAC1. c Photos of maize among various treatments. Scale bar: 1 cm. d Growth evaluation of roots by measuring lateral root number and density. Fifteen seedlings were used to collect the data. e Photos of maize ND101 (widely type) and transgenic maize overexpressing miR164e. Scale bar: 1 cm. f Growth evaluation of roots by measuring lateral root number and density. Thirteen seedlings were used to collect the data. The “n.s.” means no significance, and the “***” indicates significant differences according to the independent t test (P < 0.001)
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