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. 2021 Jun 25;9(2):32-47.
doi: 10.14252/foodsafetyfscj.D-20-00032. eCollection 2021 Jun.

Effect of Transgenic Rootstock Grafting on the Omics Profiles in Tomato

Hiroaki Kodama  1 , Taira Miyahara  1 , Taichi Oguchi  2   3 , Takashi Tsujimoto  4 , Yoshihiro Ozeki  4 , Takumi Ogawa  5 , Yube Yamaguchi  5 , Daisaku Ohta  5
Affiliations

Effect of Transgenic Rootstock Grafting on the Omics Profiles in Tomato

Hiroaki Kodama et al. Food Saf (Tokyo). .

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 Micro-Tom rootstocks. These multiple omics data showed that grafting on the rootstock harboring the GUS transgene did not induce any genetic or metabolic variation in the scion.

Keywords: Solanum lycopersicum; genetically modified (GM) plants; grafting; new plant breeding technology (NPBT); omics analysis; tomato.

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Conflict of interest statement

Conflict of Interest: The authors have no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Phenotype of non-transgenic Stella Mini Tomato (S) scions grafted on transgenic and non-transgenic Micro-Tom (T-MT and N-MT, respectively) as rootstocks. (A) Aerial parts of plant shapes of S scions grafted on respective T-MT and N-MT at the beginning of the flowering stage (mid of June in 2019). Stella scions grafted on Stella rootstocks and non-grafted Stella are shown as the control. (B, C) Phenotypes of ripe fruits settled on S scions grafted on respective T-MT (B) and N-MT (C). (D) Fruit ripening stages settled on non-grafted Stella. Fruits past 10 DAB of 7 DAB defined the fully ripened stage. (E) Fresh weight of fruits settled on Stella scions grafted on T-MT, N-MT, and Stella rootstock and non-grafted Stella harvested at 7 DAB. Different letters above the box plots indicate significant differences among the grafted plants according to the Tukey-HSD test (ɑ = 0.05). (F) Detection of transgenes for NPTII and GUS genes and CaMV 35S promoter region in the T-MT. As PCR control, a primer pair for the tomato endogenous PG gene was used. No amplified products for transgenes could be detectable in N-MT.
Fig. 2.
Fig. 2.
(A) Hierarchical cluster tree of transcripts in fruits derived from ST1, 2, and 3 lines and SN1, 2, and 3 lines. Dendrogram generated from 22,035 genes expressed at least in one of the six samples. (B) Analysis of expression patterns of DEGs. Functional annotation of each DEG and logFC (ST vs. SN) are shown for the ST and SN fruits.
Fig. 3.
Fig. 3.
Comparison of digested protein composition in fruits from grafted tomato plants. (A) PCA score plot of peptide analysis by UHPLC-ESI-MS obtained in positive mode. (B) PCA score plot of peptide analysis by UHPLC-ESI-MS obtained in and negative mode. Each plot represents an individual analytical sample. Percentage values in parentheses are the respective contribution ratios.
Fig. 4.
Fig. 4.
Comparison of metabolite composition in fruits from grafted tomato plants. (A, B) PCA score plots of metabolite analysis using LC-ESI-MS obtained in positive mode and negative mode, respectively. Each plot represents an individual analytical sample. Percentage values in parentheses are the respective contribution ratios. (C) Comparison of metabolite compositions in fruits from the grafted tomato plants using GC-EI-MS analysis. A PCA score plot of metabolite profile. Each plot represents an individual analytical sample. (D) Each plot represents an individual ion selected from each peak group. The numbers next to the plots represent their peak group ID. Tomato grafted plants, ST1, ST2, and ST3, were generated by grafting non-transgenic scions from Stella Mini Tomato onto transgenic Micro-Tom (MT) rootstocks expressing a GUS gene under the control of CaMV35S promoter. The control grafted plants, SN1, SN2 and SN3, were comprised of non-transgenic Stella scions and non-transgenic MT rootstocks. Three individual fruit samples from each grafted plant were subjected to MS analyses. Thus, three analytical results are shown for individual grafted plants (SN1-1, SN1-2, and SN1-3 from SN1 grafted plant, for example).
Fig. 5.
Fig. 5.
Contents of α-tomatine and esculeoside A in the ST and SN fruits. Relative signal intensity of peak group annotated as α-tomatine (A) and peak group predicted as esculeoside A (B). Error bars indicate standard deviation from three independent fruits from same grafted plant lines.
Fig. 6.
Fig. 6.
General food ingredients of fully ripened tomato fruits. The water, protein, lipid, ash, and carbohydrate contents of fruits settled on Stella scions grafted on T-MT and N-MT rootstocks (S/T and S/N, respectively) and Stella rootstock (S/S) and non-grafted Stella are displayed in A–E, respectively. Different letters at the above of the bars indicate significant differences among the grafted plants according to the Tukey-HSD test (ɑ = 0.05). Error bars represent standard error.
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