Daisaku Ohta

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.

Transgrafting constitutes a technique involving the integration of genetically modified (GM) and non-GM plant organisms. Typically, edible components derived from non-GM scions are categorized as non-GM food products, attributed to the absence of exogenous genetic material within their respective genomes. Non-GM food status could be compromised if proteins translocated across the graft interface. We investigated the movement of insecticidal Bacillus thuringiensis (Bt) crystal proteins, widely utilized in GM crop species. Tobacco plants engineered to express the Cry1Ab gene exhibited trace levels of Cry1Ab protein accumulation. In transgrafted plants, translocated Cry1Ab protein originating from GM rootstocks was detectable within scion foliar tissues but not within the seeds obtained from the non-GM scion. This result unequivocally demonstrates the capacity for Bt protein translocation from rootstocks to scions yet indicates a constrained distribution confined to scion tissues relatively close to the graft junction. While regulatory considerations necessitate a thorough appraisal of potential risks associated with Bt proteins, the results shown here facilitate the commercialization of the edible components as non-GM food products.

Plant viruses use the plasmodesmata and vascular systems to spread systemically in a plant, which may influence the translocation of exogenous transgene products in genetically modified (GM) plants. Transgrafting is a technique that involves the use of GM plants as grafting partners for non-GM plants, and yields non-GM edible harvests from transgrafted crops; thus, there is potential for its distribution as a non-GM product. However, when growing in agricultural fields, transgrafts are exposed to biotic stresses, such as plant virus infections. In this study, we investigated the influence of a plant virus infection on translocation of transgene products between GM and non-GM parts of transgrafts. We generated homo- and hetero-transgrafts of green fluorescent protein (GFP)-expressing GM tomatoes and GM Nicotiana benthamiana rootstocks with non-GM tomato scions and infected them with cucumber mosaic virus (CMV), a major plant virus, and analyzed the translocation of GFP protein in transgrafts. The results showed that CMV infection did not promote GFP transfer from GM rootstock to non-GM scions.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

Grafting has been widely applied in agricultural production in order to utilize agriculturally valuable traits. The use of genetically modified (GM) plants for grafting with non-GM crops will soon be implemented to generate chimeric plants (transgrafting)*, and the non-GM edible portions thus obtained could fall outside of the current legal regulations. A number of metabolites and macromolecules are reciprocally exchanged between scion and rootstock, affecting the crop properties as food. Accordingly, the potential risks associated with grafting, particularly those related to transgrafting with GM plants, should be carefully evaluated based on scientific evidence. In this study, we prepared a hetero-transgraft line composed of non-GM tomato scion and GM-tobacco rootstock expressing firefly luciferase. We also prepared a homograft line (both rootstock and scion are from non-GM tomato) and a heterograft line (non-GM tobacco rootstock and non-GM tomato scion). The non-GM tomato fruits were harvested from these grafted lines and subjected to comprehensive characterization by multi-omics analysis. Proteomic analysis detected tobacco-derived proteins from both heterograft and hetero-transgraft lines, suggesting protein transfer from the tobacco rootstock to the tomato fruits. No allergenicity information is available for these two tobacco-derived proteins. The transcript levels of the genes encoding two allergenic tomato intrinsic proteins (Sola l 4.0101 and Sola l 4.0201) decreased in the heterograft and hetero-transgraft lines. Several differences were observed in the metabolic profiles, including α-tomatine and nicotine. The accumulation of tobacco-derived nicotine in the tomato fruits of both heterograft and hetero-transgraft lines indicated that the transfer of unfavorable metabolites from rootstock to scion should be assessed as a food safety concern. Further investigations are needed to clarify whether variable environmental conditions and growth periods may influence the qualities of the non-GM edible parts produced by such transgrafted plants.

UV-C irradiation increases resveratrol content in grape skins, but it reaches a maximum at a certain UV-C dose. In contrast, UV-B has a weak resveratrol-enhancing effect at low doses, but it has not been investigated at high doses. In this study, we investigated the effect of high-dose UV-B on resveratrol contents in grape skins. Irradiation of Muscat Bailey A with 290 nm UV-B LED at 22 500 and 225 000 µmol m⁻² increased the resveratrol contents in the grape skins by 2.1- and 9.0-fold, respectively, without significant increases in other phenolic compounds. The effect was also confirmed for 2 other cultivars: Shine Muscat and Delaware. Transcriptome analysis of the grape skins of Muscat Bailey A immediately after irradiation with UV-B at 225 000 µmol m⁻² showed that genes related to biotic and abiotic stresses were upregulated. Hence, it was suggested that high-dose UV-B irradiation induces a stress response and specifically activates resveratrol biosynthesis.

Root parasitic weeds of the Orobanchaceae, such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), cause serious losses in agriculture worldwide, and efforts have been made to control these parasitic weeds. Understanding the characteristic physiological processes in the life cycle of root parasitic weeds is particularly important to identify specific targets for growth modulators. In our previous study, planteose metabolism was revealed to be activated soon after the perception of strigolactones in germinating seeds of O. minor. Nojirimycin inhibited planteose metabolism and impeded seed germination of O. minor, indicating a possible target for root parasitic weed control. In the present study, we investigated the distribution of planteose in dry seeds of O. minor by matrix-assisted laser desorption/ionization–mass spectrometry imaging. Planteose was detected in tissues surrounding—but not within—the embryo, supporting its suggested role as a storage carbohydrate. Biochemical assays and molecular characterization of an α-galactosidase family member, OmAGAL2, indicated that the enzyme is involved in planteose hydrolysis in the apoplast around the embryo after the perception of strigolactones, to provide the embryo with essential hexoses for germination. These results indicate that OmAGAL2 is a potential molecular target for root parasitic weed control.

Root parasitic weeds of the Orobanchaceae, such as witchweeds ( Striga spp.) and broomrapes ( Orobanche and Phelipanche spp.), cause serious losses in agriculture worldwide. No practical method to control these parasitic weeds has been developed to date. Understanding the characteristic physiological processes in the life cycles of root parasitic weeds is particularly important to identify specific targets for growth modulators. In our previous study, planteose metabolism was revealed to be activated soon after the perception of strigolactones in germinating seeds of O . minor . Nojirimycin inhibited planteose metabolism and impeded seed germination of O . minor , indicating that planteose metabolism is a possible target for root parasitic weed control. In the present study, we investigated the distribution of planteose in dry seeds of O . minor by matrix-assisted laser desorption/ionization–mass spectrometry imaging. Planteose was detected in tissues surrounding—but not within—the embryo, supporting its suggested role as a storage carbohydrate. Biochemical assays and molecular characterization of an α-galactosidase family member, OmAGAL2, indicated the enzyme is involved in planteose hydrolysis in the apoplast around the embryo after the perception of strigolactones to provide the embryo with essential hexoses for germination. These results indicated that OmAGAL2 is a potential molecular target for root parasitic weed control. Highlight: Planteose accumulated in tissues surrounding the embryo in Orobanche minor dry seeds and was indicated to be hydrolyzed in the apoplast around the embryo by α-galactosidase during germination.

SUMMARY: In plants, the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) perceives UV-B and induces UV-B responses including synthesis of UV-B absorbing phenolic compounds such as anthocyanins. UVR8 absorbs a range of UV-B (260–335 nm). However, the responsiveness of plants to each UV-B wavelength has not been intensively studied so far. Here, we performed transcriptome and metabolome analyses of Arabidopsis using UV light emitting diodes (LEDs) with peak wavelengths of 280 and 310 nm to investigate the differences in the wavelength-specific UV-B responses. Irradiation with both UV-LEDs induced gene expression of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which has a central role in the UVR8 signaling pathway. However, the overall transcriptomic and metabolic responses to 280 and 310 nm UV-LED irradiation were different. Most of the known UV-B-responsive genes, such as salicylic acid, jasmonic acid, and defense-related genes, responded only to 280 nm UV-LED irradiation. Lipids, polyamines and organic acids were the metabolites most affected by 280 nm UV-LED irradiation, whereas the effect of 310 nm UV-LED irradiation on the metabolome was considerably less. Enzymatic genes involved in the phenylpropanoid pathway upstream in anthocyanin biosynthesis were up-regulated only by 280 nm UV-LED irradiation. On the other hand, no enzymatic genes downstream in anthocyanin biosynthesis were induced by the UV-LEDs, but rather, they were down-regulated by 310 nm UV-LED irradiation. These results revealed that the responsivenesses of Arabidopsis to 280 and 310 nm UV-B were significantly different, suggesting that UV-B signaling is mediated by more complex pathways than the current model.