Omics Profiles of the Null Segregants of RNA-Directed DNA Methylation-Positive Tobacco Plants

RNA-directed DNA methylation (RdDM), a new plant breeding technology, induces epigenetic modifications that can be inherited even after segregation of the responsible transgene. The transgene-free descendants (null segregants) are potentially exempt from the regulation of genetically modified plants. To evaluate the risks of potential unintended molecular changes in the null segregants of RdDM-positive plants, we produced null segregants (S44end2-null) from a transgenic tobacco line in which RdDM targeting the promoter of the transgene was introduced. Comprehensive multi-omics analyses, including transcriptomics, proteomics, and metabolomics, were conducted using S44end2-null and wild-type (WT) plants. Principal component analysis demonstrated clear separation of the transcriptomic and proteomic profiles of the two groups. The metabolomic profiles of S44end2-null plants exhibited considerable overlap with those of WT plants. Proteomic analysis of the null segregants of tobacco plants transformed with an empty vector demonstrated distinct cluster separation from WT plants. Because only sporadic DNA methylation on the tobacco genome was expected by the RdDM construct used in this study, the observed differences in omics profiles are considered to be significantly influenced by genetic variation accumulated during the transformation and regeneration processes (somaclonal variation). The safety assessment points for null segregants using RdDM technology are discussed.