46 | December 2021 www.facs.website Gene delivery is a powerful biotechnology for engineering plants based on modifying plant organelles (nucleus, chloroplasts, and mitochondria) to acquire desired traits for material production. To date, plant modification has been mainly achieved via agrobacterium-mediated or biolistic transformation, which suffers from several limitations for plant species and organelles. We have utilized functional polypeptides synthesized by chemoenzymatic polymerization as tools to modify plants (Figure 5). Exogenous genes, in the form of plasmid DNA (pDNA), double-stranded DNA (dsDNA), or RNA, electrostatically interact with a cationic peptide carrier to form a peptide/DNA complex.16 The peptide carrier consists of two or more functional sequences, including cationic DNA condensing domains, cell-penetrating peptides (CPPs), and organellar transit peptides, to efficiently translocate cargoes into plant cells. The peptide/DNA complex is typically delivered into plant cells to engineer organelles by immersing plants (tissues) into the complex solution or direct infiltration using a syringe. We have rationally designed functional polypeptides that individually match requirements for overcoming barriers for internalization into target organelles that we aim to modify. For this purpose, chemoenzymatic synthesis is a useful technique to synthesize such functional sequences. As a sophisticated carrier that enables DNA cargoes to enter plant cells, we developed lysine-based cationic peptides ligated with a terminal-functionalized oligo(ethylene glycol) (Figure 6).17 By mixing the cationic peptide with DNA, a micellar complex immediately forms by electrostatic interaction, and the resulting micelle complex exhibits reactive functional groups such as maleimide and alkyne on its surface. We can exploit the surface reactive groups to further modify the micelle complex with another functional peptide or a peptide cocktail with multiple functionalities. The thiol-maleimide click reaction using cysteine-terminated functional peptides easily postfunctionalizes the micelle complex at the surface after the complexation process, and the types of functions and the reaction degree can be tuned on demand. Our fundamental experiments reveal that postfunctionalization with CPP doubles the gene delivery efficiency of a functionalized peptide/DNA complex compared with a nonfunctionalized micelle complex in a model plant (Arabidopsis thaliana). Fur thermore, when the complex is modified with two peptides, CPP and endosome disrupting peptide (EDP), at an optimized ratio of 1 to 1, the gene delivery ef ficiency is further increased.18 The doubly functionalized complex wi th CPP and EDP can ef fectively deliver the DNA cargo into cel ls via CPP-mediated endocytosis and subsequently release it from the endosome to the cytosol. This success in improving delivery efficiency strongly implies that multiple functionalizations to address each barrier during the internalization process are essential to achieve high gene delivery efficiency. The most important function for internalization into cells is the membrane penetration property. CPPs have been developed as biological tools to deliver biomolecules into cells, initially for animal cells. We screened known CPPs originally used for material delivery into animal cells for internalization into plant cells to assess their availability for plant modification.19 Among the three categories of CPPs, namely, cationic, hydrophobic, and amphiphilic peptides, some amphiphilic CPPs were found to show a high internalization ability for model plant cells, although we found no remarkable correlation between the type/sequence of CPPs and the plant species that was used. In particular, no CPP was found that could be adapted to all plant species. The screening results for the CPP library motivated us to develop a novel artificial CPP suitable for versatile use across all plant species and tissue types. The most suitable candidate CPP in the peptide library was found to be an amphiphilic peptide adopting a helical conformation, which is assumed to be the key feature to penetrate cell membranes. We attempted to introduce a bulky nonproteinogenic Aib Figure 7. Zwitterionic polypeptides for dissociation of cellulose networks in cell walls. High-speed atomic force microscopy reveals that polypeptide treatment of cultured plant cells leads to partial dissociation of the cellulose network and amorphous pectin layer in cell walls. Bars show 50 nm. Reproduced from Ref. 23 with permission from the American Chemical Society. Figure 6. Infiltration of the peptide/DNA micelle complex into plant cells. The micelle complex, which displays reactive functional groups (maleimides) on its surface, can be postfunctionalized with various functional peptides, including cell penetrating peptides and organellar transit peptides, via a click reaction.
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