Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

Angiosperm mitochondrial genomes have highly complex and diverse structures that are partly due to frequent insertions of nuclear and chloroplast DNA (cpDNA) into mitochondrial DNA (mtDNA). This suggests the existence of mechanisms for gene transfer from chloroplasts to mitochondria, but these have yet to be discovered. In this study, we aimed to detect chloroplast-to-mitochondrion gene transfer by analyzing the translocation of a marker gene, sul, encoding a bacterial dihydropteroate synthase that confers sulfonamide resistance in tobacco (Nicotiana tabacum), to mtDNA. First, we created tobacco chloroplast transformants in which sul, surrounded on both sides by ~ 1 kb of mitochondrial homologous sequences that enable targeted integration into mtDNA, was introduced into the chloroplast genome. Heat shock enhanced sul expression in the transformants, suggesting that chloroplast degradation stimulates gene transfer from chloroplasts to mitochondria. Shoot regeneration using the heat-shocked chloroplast transformants under sulfadiazine selection resulted in several transformants showing moderately resistant to sulfadiazine. Deep sequencing analysis of the target mitochondrial locus detected sul in the SR plants with an integration efficiency of 0.0007–0.0036%, and we validated the results by ruling out two types of artifactual outcomes, PCR jumping and sul integration into nuclear mitochondrial DNA (NuMT). From these results, we propose that gene transfer from chloroplasts to mitochondria is ongoing in tobacco.