Effect of Transgenic Rootstock Grafting on the Omics Profiles in Tomato

Abstract

Grafting of non-transgenic scion onto genetically modified (GM) rootstocks provides superior agronomic traits in the GM rootstock, and excellent fruits can be produced for consumption. In such grafted plants, the scion does not contain any foreign genes, but the fruit itself is likely to be influenced directly or indirectly by the foreign genes in the rootstock. Before market release of such fruit products, the effects of grafting onto GM rootstocks should be determined from the perspective of safety use. Here, we evaluated the effects of a transgene encoding β-glucuronidase (GUS) on the grafted tomato fruits as a model case. An edible tomato cultivar, Stella Mini Tomato, was grafted onto GM Micro-Tom tomato plants that had been transformed with the GUS gene. The grafted plants showed no difference in their fruit development rate and fresh weight regardless of the presence or absence of the GUS gene in the rootstock. The fruit samples were subjected to transcriptome (NGS-illumina), proteome (shotgun LC-MS/MS), metabolome (LC-ESI-MS and GC-EI-MS), and general food ingredient analyses. In addition, differentially detected items were identified between the grafted plants onto rootstocks with or without transgenes (more than two-fold). The transcriptome analysis detected approximately 18,500 expressed genes on average, and only 6 genes were identified as differentially expressed. Principal component analysis of 2,442 peaks for peptides in proteome profiles showed no significant differences. In the LC-ESI-MS and GC-EI-MS analyses, a total of 93 peak groups and 114 peak groups were identified, respectively, and only 2 peak groups showed more than two-fold differences. The general food ingredient analysis showed no significant differences in the fruits of Stella scions between GM and non-GM

Omics Profiles of Non-transgenic Scion Grafted on Transgenic RdDM Rootstock

“…The metabolome was analyzed using high performance liquid chromatography (LC)-ESI-MS and gas chromatography (GC)-electron ionization (EI)-MS following a previously reported protocol 24) .…”

“…The supernatant was filtered through a 0.2-µm PTFE membrane (Millex-LG; Merck Millipore, Billerica, MA). Nontargeted metabolite analysis was carried out by LC-ESI-MS using a LCMS-8040 system (Shimadzu Corp., Kyoto, Japan) as described previously 24) . Mass spectra within the m/z range of 100-1000 were obtained by Q3 scan mode with positive/ negative polarity switching.…”

“…NPBTs include the genome editing, oligo-directed mutagenesis, cisgenesis and transgenesis, RdDM, and grafting 23) . In a previous paper, we showed that a transgenic protein (β-glucuronidase) expressed in GM tomato rootstock did not cause unintended effects on transcriptomic, proteomic, or metabolomic traits affecting food safety in the non-GM scion 24) . Here, to evaluate the risk of grafting techniques involving epigenetic regulation, we investigated the effects of RdDM-inducing tobacco rootstock on a non-GM scion (i.e., as a model testing the long-distance movement of siRNAs).…”

Recent applications of metabolomics in plant breeding

“… Chevalier et al (2021) evaluated phenotypic plasticity in carrot varieties and concluded that a balance between constitutive content and the environmental sensitivity of key metabolites should be considered for quality improvement in carrot and other vegetables. For the assessments of the unintended effects of genetically modified crops, reports have been published on maize varieties ( Liu et al 2021 ) and tomato rootstock ( Kodama et al 2021 ).…”

Proteomics of Reproductive Development, Fruit Ripening, and Stress Responses in Tomato

“…The variation in most of the proteins identified, mainly those involved in translation, stress, cytoskeleton rearrangement, folding, and enzymes from biosynthetic pathways, were biologically insignificant according to mean statistical analysis (ANOVA), suggesting that the GM plants were substantially equivalent to the parent lines and were safe for consumption . Even the evaluation of the rootstocks of a transgenic tomato grafted with a nontransgenic scion by the omics approach showed that fruit quality improved without the presence of a transgene in the grafted tomato plant . There was no metabolic or genetic variations in the grafted stock that would result in a cause of concern for consumers.…”