Acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana (Arabidopsis) accessions. Most salt-tolerant accessions exhibit acquired osmotolerance, whereas Col-0 does not. To identify genes that can confer acquired osmotolerance to Col-0 plants, we performed full-length cDNA overexpression (FOX) hunting using full-length cDNAs of halophyte Eutrema salsugineum, a close relative of Arabidopsis. We identified EsCYP78A5 as a gene that can confer acquired osmotolerance to Col-0 wild-type (WT) plants. EsCYP78A5 encodes a cytochrome P450 monooxygenase and the Arabidopsis ortholog is known as KLU. We also demonstrated that transgenic Col-0 plants overexpressing AtKLU (AtKLUox) exhibited acquired osmotolerance. Interestingly, KLU overexpression improved not only acquired osmotolerance but also osmo-shock, salt-shock, oxidative, and heat-stress tolerances. Under normal conditions, the AtKLUox plants showed growth retardation with shiny green leaves. The AtKLUox plants also accumulated higher anthocyanin levels and developed denser cuticular wax than WT plants. Compared to WT plants, the AtKLUox plants accumulated significantly higher levels of cutin monomers and very-long-chain fatty acids, which play an important role in the development of cuticular wax and membrane lipids. Endoplasmic reticulum (ER) stress induced by osmotic or heat stress was reduced in AtKLUox plants compared to WT plants. These findings suggest that KLU is involved in the cuticle biosynthesis, accumulation of cuticular wax, and reduction of ER stress induced by abiotic stresses, leading to the observed abiotic stress tolerances.
Rapid DNA template preparation directly from a single rice (Oryza sativa) grain or rice flour of its equivalent weight was developed for loop-mediated isothermal amplification (LAMP). LAMP efficiency using DNA extract obtained from consecutive addition of alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) and neutralizing reagent (40 mM Tris–HCl [pH 5]) was comparable to that using an equivalent amount of purified DNA as template. The stability of the prepared DNA extract was confirmed for up to six-day storage at room temperature. Without using any special laboratory devices, the developed method enabled a rapid, simple, and low-cost DNA template preparation method for reliable LAMP testing to detect rice genes.
Many countries have implemented the labeling system of genetically modified organisms (GMO). In Japan, the regulatory threshold for non-GMO labeling will be revised and restricted to undetectable by April 2023. The practical criterion for the revised system is based on the limit of detection (LOD). However, determining whether the commingling of GMO levels exceeds the LOD is challenging because GM contents close to the LOD are usually below the limit of quantification. In this study, we developed a qualitative method based on comparative Cq-based analysis targeting cauliflower mosaic virus 35S promoter and GM soybean MON89788 event-specific sequences that could be applicable to the revised non-GMO labeling. ΔCq values between the target and endogenous sequences were calculated, and the ΔΔCq value obtained was used as a criterion to determine analytical samples with GM contents exceeding the threshold. To improve the reproducibility of the method, we used a standard plasmid that yields equivalent and stable ΔCq values comparable with those obtained from LOD samples. The developed method was validated with an interlaboratory study. The new qualitative detection concept would be useful for ensuring robust and reproducible results among laboratories, particularly for detecting low-copy-number DNA samples.
A genetically modified (GM) soybean kernel detection system using combination of DNA preparation from individual soybean kernels and event-specific real-time PCR was developed to simultaneously identify GM soybean events authorized for food after safety assessments in Japan. Over 100 kernels in the nonidentity-preserved soybean samples imported from the United States of America (two U.S.A. lots) and Brazil (one lot) were randomly selected and examined. In total, 98 and 96% of the two independent U.S.A. lots, and 100% of the Brazilian lot contained GM soybean kernels. Herbicide-tolerant events, MON89788 (trade name Genuity ® Roundup Ready 2 Yield), GTS 40-3-2 (trade name Roundup Ready soybean) and A2704-12 (trade name Liberty Link ® soybean), were detected similarly in both U.S.A. lots. In the Brazilian lot, in addition to GTS 40-3-2, a stacked GM event, MON87701 MON89788, having insect-resistance and herbicidetolerance, was detected. There were no unauthorized GM soybeans comingled, and the ratio of GM soybean events detected was consistent with statistical reports on the cultivated GM soybean events in both countries.
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