Targeted Gene Delivery into Various Plastids Mediated by Clustered Cell‐Penetrating and Chloroplast‐Targeting Peptides

Abstract

The plastid is an organelle that functions as a cell factory to supply food and oxygen to the plant cell and is therefore a potential target for genetic engineering to acquire plants with novel photosynthetic traits or the ability to produce valuable biomolecules. Conventional plastid genome engineering technologies are laborious for the preparation of plant material, require expensive experimental instruments, and are time consuming for obtaining a transplastomic plant line that produces significant levels of the biomolecule of interest. Herein, a transient plastid transformation technique is presented using a peptide‐based gene carrier. By formulating peptide/plasmid DNA complexes that combine the functions of both a cell‐penetrating peptide and a chloroplast‐targeting peptide, DNA molecules are translocated across the plant cell membrane and delivered to the plastid efficiently via vesicle formation and intracellular vesicle trafficking. A simple infiltration method enables the introduction of a complex solution into intact plants, and plastid‐localized transgene expression is expeditiously observed in various types of plastids in differentiated cell types of several plants. The gene delivery technology thus provides a useful tool to rapidly engineer plastids in crop species.

Simultaneous introduction of multiple biomacromolecules into plant cells using a cell-penetrating peptide nanocarrier

“…57,58 In addition, biomolecules can be precisely targeted to plastids, mitochondria, peroxisomes and vacuoles using a combination of functionalized CPPs and organelle-targeting peptides (OTPs). [59][60][61] These CPP/OTP-based carriers facilitate effective reprogram-ming of organellar metabolic pathways in crop species. However, major drawbacks of such peptide-based biomolecule delivery systems are the production costs of the peptides and biomolecules.…”

“…The CPP-based multiple-biomolecule delivery system is compatible with different plant species and various biomolecules of interest but does not require specialized equipment or complicated plant material preparation. Furthermore, the CPP can be combined with selected OTPs, i.e., peptides that designate biomolecule translocation to specific organelles, 60,61 for targeted delivery of multiple components to various plant organelles. Our peptide-mediated multiple-biomolecule delivery platform provides a robust tool for comprehensive engineering of plant metabolic processes and quality trait improvement.…”

Enhancers in polymeric nonviral gene delivery systems

“…Numata's group reported a peptide‐based gene delivery system for DNA delivery targeting plastids (Figure 5). 54 The pDNA/CTP/CPP complex formed when the N/P ratio is 1.0 and 2.5 maximized Rluc activity (2.1 times) compared with the pDNA/CTP complex alone. Reineke's group found that adding heparin to the glycopolymer/pDNA polyplexes after complexation enhanced gene delivery 55 .…”

Species-independent analytical tools for next-generation agriculture

“…It has been shown that the zeta potential and size of nanoparticles govern their subcellular localization within plant cells, enabling the specific targeting of various compartments such as the chloroplasts and the cytosol 75,112,113 . Furthermore, targeting moieties can be grafted onto the surface of nanostructures to direct their localization to the nucleus, mitochondria or other organelles within a plant cell 114,115 . While these strategies can be rationally employed to control nanoparticle transport into specific organelles, biomolecules present in the plant biological environment can rapidly adsorb onto nanoparticle surfaces in an energetically favourable process and alter the nanoparticle surface characteristics 116,117 .…”