Abstract
Grafting of commercial varieties onto transgenic stress-tolerant rootstocks is attractive approach, because fruit from the non-transgenic plant body does not contain foreign genes. RNA silencing can modulate gene expression and protect host plants from viruses and insects, and small RNAs (sRNAs), key molecules of RNA silencing, can move systemically. Here, to evaluate the safety of foods obtained from sRNA-recipient plant bodies, we investigated the effects of rootstock-derived sRNAs involved in mediating RNA-directed DNA methylation (RdDM) on non-transgenic scions. We used tobacco rootstocks showing RdDM against the cauliflower mosaic virus (CaMV) 35S promoter. When scions harboring CaMV 35S promoter sequence were grafted onto RdDM-inducing rootstocks, we found that RdDM-inducing sRNAs were only weakly transported from the rootstocks to the scion, and we observed a low level of DNA methylation of the CaMV 35S promoter in the scion. Next, wild-type (WT) tobacco scions were grafted onto RdDM-inducing rootstocks (designated NT) or WT rootstocks (designated NN), and scion leaves were subjected to multi-omics analyses. Our transcriptomic analysis detected 55 differentially expressed genes between the NT and NN samples. A principal component analysis of proteome profiles showed no significant differences. In the positive and negative modes of LC-ESI-MS and GC-EI-MS analyses, we found a large overlap between the metabolomic clusters of the NT and NN samples. In contrast, the negative mode of a LC-ESI-MS analysis showed separation of clusters of NT and NN metabolites, and we detected 6 peak groups that significantly differed. In conclusion, we found that grafting onto RdDM-inducing rootstocks caused a low-level transmission of sRNAs, resulting in limited DNA methylation in the scion. However, the causal relationships between sRNA transmission and the very slight changes in the transcriptomic and metabolomic profiles of the scions remains unclear. The safety assessment points for grafting with RdDM rootstocks are discussed.
Highlights
Many species of fruit trees including peach, apple, and persimmon are grafted onto a rootstock to confer improved resistance against environmental stresses, thereby enabling rapid propagation of elite cultivars by farmers
To determine whether siRNAs produced in the S44-end[2] rootstocks were able to induce RNAdirected DNA methylation (RdDM) in the scion, we used LUC plants as scions. This genetically modified (GM) scion harbors a transgene consisting of CaMV 35S promoter and luciferase cDNA
We were not able to construct a good discriminant model using these two groups, which suggests that there is no significant difference between them. These results suggest that the expression of RdDM-inducing siRNAs in transgenic tobacco rootstocks did not have a significant effect on the translated products of non-GM tobacco scions
Summary
One parental plant with superior agricultural traits for abiotic and/or biotic stress, such as drought, salinity, abnormal temperature tolerance, and resistances against plant pests and pathogens such as viruses, fungi and insects, is crossed with another parental plant that is less tolerant but possesses superior genomic traits of features linked to consumer preference, such as good nutrition, taste and yield To combine these traits into a single plant cultivar or variety, numerous and repeated rounds of hybridizing and backcrossing, which involves tedious labor in the field, is required. The transgenes are not introduced into the scion part and the fruits ripening on the scions should be non-GM foods
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