Development and Performance Evaluation of a New Test Kit for Quantifying the Degree of DNA Fragmentation.

BACKGROUND: PCR-based genetic testing of agricultural products and foods is widely used for detecting various analytical targets such as genetically modified organisms and food allergens. However, it is difficult to obtain accurate genetic testing results from processed foods because DNA is fragmented by heat and pressure during food processing. Thus, we previously developed an analytical method to quantitatively evaluate the degree of DNA fragmentation for the purpose of QC of genetic testing for processed foods. OBJECTIVE: Our previous analytical method requires four PCR primer sets, resulting in high reagent costs and heavy analytical workloads. Therefore, we attempted to develop an easy-to-use test kit for quantifying the degree of DNA fragmentation and to evaluate its analytical performance. METHODS: To simplify the analysis procedure, we used only two primer sets. In addition, no-fragmentation control templates were prepared to obtain stable measurement results. The precision of the simplified analysis was evaluated through blind tests between laboratories. RESULTS: It was confirmed that plant species and extracted DNA concentrations had little effect on analysis with the newly developed test kit. In addition, the analytical values indicating the degree of DNA fragmentation exhibited small variability between laboratories. CONCLUSION: We confirmed the high practicality of the developed test kit. Because DNA fragmentation in cells is a universal phenomenon, we anticipate that the test kit will be used not only for QC of genetic testing but also for food testing, medical diagnostics, and other applications in a range of fields. HIGHLIGHTS: The newly developed test kit enables quantitative evaluation of the degree of DNA fragmentation in a simple manner.

Extracellular oil production by Rhodotorula paludigena BS15 for biorefinery without complex downstream processes.

To realize biomass refinery without complex downstream processes, we extensively screened for microbial strains that efficiently produce extracellular oil from sugars. Rhodotorula paludigena (formerly Rhodosporidium paludigenum) BS15 was found to efficiently produce polyol esters of fatty acids (PEFAs), which mainly comprised of 3-acetoxypalmitic acid and partially acetylated mannitol/arabinitol. To evaluate the performance of this strain, fed-batch fermentation was demonstrated on a flask scale, and 110 g/L PEFA and 103 g/L dry cells were produced in 12 days. To the best of our knowledge, the strain BS15 exhibited the highest PEFA titer (g/L) ever to be reported so far. Because the PEFA precipitated at the bottom of the culture broth, it could be easily recovered by simply discarding the upper phase. Various carbon sources can be utilized for cell growth and/or PEFA production, which signifies the potential for converting diverse biomass sources. Two different types of next-generation sequencers, Illumina HiSeq and Oxford Nanopore PromethION, were used to analyze the whole-genome sequence of the strain BS15. The integrative data analysis generated a high-quality and reliable reference genome for PEFA-producing R. paludigena. The 22.5-M base genome sequence and the estimated genes were registered in Genbank (accession numbers BQKY01000001-BQKY01000019). KEY POINTS: • R. paludigena BS15 was isolated after an extensive screening of extracellular oil producers from natural sources. • Fed-batch fermentation of R. paludigena BS15 yielded 110 g/L of PEFA, which is the highest titer ever reported to date. • Combined analysis using Illumina and Oxford Nanopore sequencers produced the near-complete genome sequence.

Development and Validation of a New Robust Detection Method for Low-Content DNA Using ΔΔCq-Based Real-Time PCR with Optimized Standard Plasmids as a Control Sample.

Real-time polymerase chain reaction (PCR) is the gold standard for DNA detection in many fields, including food analysis. However, robust detection using a real-time PCR for low-content DNA samples remains challenging. In this study, we developed a robust real-time PCR method for low-content DNA using genetically modified (GM) maize at concentrations near the limit of detection (LOD) as a model. We evaluated the LOD of real-time PCR targeting two common GM maize sequences (P35S and TNOS) using GM maize event MON863 containing a copy of P35S and TNOS. The interlaboratory study revealed that the LOD differed among laboratories partly because DNA input amounts were variable depending on measurements of DNA concentrations. To minimize this variability for low-content DNA samples, we developed ΔΔCq-based real-time PCR. In this study, ΔCq and ΔΔCq are as follows: ΔCq = Cq (P35S or TNOS) - Cq (SSIIb; maize endogenous gene), ΔΔCq = ΔCq (analytical sample) - ΔCq (control sample at concentrations near the LOD). The presence of GM maize was determined based on ΔΔCq values. In addition, we used optimized standard plasmids containing SSIIb, P35S, and TNOS with ΔCq equal to the MON863 genomic DNA (gDNA) at concentrations near the LOD as a control sample. A validation study indicated that at least 0.2% MON863 gDNA could be robustly detected. Using several GM maize certified reference materials, we have demonstrated that this method was practical for detecting low-content GM crops and thus for validating GM food labeling. With appropriate standards, this method would be applicable in many fields, not just food.

Histidine-Containing Dipeptides Mitigate Salt Stress in Plants by Scavenging Reactive Carbonyl Species.

Reactive oxygen species (ROS) are critical factors that cause damage in salt-stressed plants, but their mechanisms of action in living cells are largely unknown. We investigated the roles of reactive carbonyl species (RCS), i.e., the lipid peroxide-derived α,ß-unsaturated aldehydes and ketones, in plant growth retardation under salt stress. When Arabidopsis thaliana Col-0 seeds were exposed to 100 mM NaCl, germination was delayed and the levels of ROS, RCS, and protein carbonylation in the seedlings were increased. Adding the histidine-containing dipeptides carnosine, N-acetylcarnosine, and anserine, which are reported RCS scavengers, restored the germination speed and suppressed the increases in RCS and protein carbonylation but did not affect the ROS level. Increases in the levels of the RCS acrolein, crotonaldehyde, (E)-2-pentenal, and 4-hydroxy-(E)-2-nonenal were positively correlated with the delay of germination and growth inhibition. These RCS, generated downstream of ROS, are thus primarily responsible for the salt-stress symptoms of plants.

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan.

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.

Reactive Carbonyl Species Inhibit Blue-Light-Dependent Activation of the Plasma Membrane H+-ATPase and Stomatal Opening.

Reactive oxygen species (ROS) play a central role in plant responses to biotic and abiotic stresses. ROS stimulate stomatal closure by inhibiting blue light (BL)-dependent stomatal opening under diverse stresses in the daytime. However, the stomatal opening inhibition mechanism by ROS remains unclear. In this study, we aimed to examine the impact of reactive carbonyl species (RCS), lipid peroxidation products generated by ROS, on BL signaling in guard cells. Application of RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), inhibited BL-dependent stomatal opening in the epidermis of Arabidopsis thaliana. Acrolein also inhibited H+ pumping and the plasma membrane H+-ATPase phosphorylation in response to BL. However, acrolein did not inhibit BL-dependent autophosphorylation of phototropins and the phosphorylation of BLUE LIGHT SIGNALING1 (BLUS1). Similarly, acrolein affected neither the kinase activity of BLUS1 nor the phosphatase activity of protein phosphatase 1, a positive regulator of BL signaling. However, acrolein inhibited fusicoccin-dependent phosphorylation of H+-ATPase and stomatal opening. Furthermore, carnosine, an RCS scavenger, partially alleviated the abscisic-acid- and hydrogen-peroxide-induced inhibition of BL-dependent stomatal opening. Altogether, these findings suggest that RCS inhibit BL signaling, especially H+-ATPase activation, and play a key role in the crosstalk between BL and ROS signaling pathways in guard cells.

Determination of Reactive Carbonyl Species, Which Mediate Reactive Oxygen Species Signals in Plant Cells.

Responses of plant cells to reactive oxygen species (ROS), e.g., reprogramming of defense genes or progression of cell death, should include the ROS signal transmission to target proteins, but the biochemistry of this process is largely unknown. Lipid peroxide-derived α,ß-unsaturated aldehydes and ketones (reactive carbonyl species; RCS), downstream products of ROS stimuli, are recently emerging endogenous agents that can mediate ROS signal to proteins via covalent modification. The involvement of RCS in certain ROS signaling in plants (oxidative injury of leaves and roots, ROS-induced programmed cell death, senescence, and abscisic acid and auxin signaling) has been verified by the determination of RCS with the use of conventional HPLC. Because distinct kinds of RCS act differently in the cell and so are metabolized, identification and quantification of each RCS in plant tissues provide central information to decipher biochemical mechanisms of plant responses to ROS. This article illustrates practical methods of plant sample preparation and extraction and analysis of RCS.

Simple, Precise, and Less Biased GMO Quantification by Multiplexed Genetic Element-Specific Digital PCR.

BACKGROUND: To provide the consumer with choices of genetically modified organisms (GMO) or non-GMO, official food labeling systems were established in many countries. Because the threshold GMO content values were set to distinguish between "non-GMO" and "GMO" designations, GMO content quantification methods are required for ensuring the appropriateness of labeling. OBJECTIVE: As the number of GMOs is continuously increasing around the world, we set out to develop a low-cost, simple and less biased analytical strategy to cover all necessary detection targets. METHODS: Digital PCR methods are advantageous compared to the conventional quantitative real-time PCR methods. We developed a digital PCR-based GMO quantification method to evaluate the GMO content in maize grains. To minimize the analytical workload, we adopted multiplex digital PCR targeting the 35S promoter and the nopaline synthase terminator, which are genetic elements commonly introduced in many GMOs. RESULTS: Our method is significantly simpler and more precise than the conventional real-time PCR-based methods. Additionally, we found that this method enables quantification of the copy number of GMO DNA without double counting multiple elements (35S promoter and nopaline synthase terminator) tandemly placed in a recombinant DNA construct. CONCLUSION: This is the first report on the development of a genetically modified maize quantification method using a multiplexed genetic element-specific digital PCR method. The tandem effect we report here is quite useful for reducing the bias in the analytical results. HIGHLIGHTS: Multiplexed genetic element-specific digital PCR can simplify weight-based GMO quantification and thus should prove useful in light of the continuous increase in the number of GM events.

Lipid Peroxide-Derived Reactive Carbonyl Species as Mediators of Oxidative Stress and Signaling.

Oxidation of membrane lipids by reactive oxygen species (ROS) or O2/lipoxygenase leads to the formation of various bioactive compounds collectively called oxylipins. Reactive carbonyl species (RCS) are a group of oxylipins that have the α,ß-unsaturated carbonyl structure, including acrolein and 4-hydroxy-(E)-2-nonenal. RCS provides a missing link between ROS stimuli and cellular responses in plants via their electrophilic modification of proteins. The physiological significance of RCS in plants has been established based on the observations that the RCS-scavenging enzymes that are overexpressed in plants or the RCS-scavenging chemicals added to plants suppress the plants' responses to ROS, i.e., photoinhibition, aluminum-induced root damage, programmed cell death (PCD), senescence, abscisic acid-induced stomata closure, and auxin-induced lateral root formation. The functions of RCS are thus a key to ROS- and redox-signaling in plants. The chemical species involved in distinct RCS signaling/damaging phenomena were recently revealed, based on comprehensive carbonyl determinations. This review presents an overview of the current status of research regarding RCS signaling functions in plants and discusses present challenges for gaining a more complete understanding of the signaling mechanisms.

Arabidopsis aldehyde oxidase 3, known to oxidize abscisic aldehyde to abscisic acid, protects leaves from aldehyde toxicity.

The Arabidopsis thaliana aldehyde oxidase 3 (AAO3) catalyzes the oxidation of abscisic aldehyde (ABal) to abscisic acid (ABA). Besides ABal, plants generate other aldehydes that can be toxic above a certain threshold. AAO3 knockout mutants (aao3) exhibited earlier senescence but equivalent relative water content compared with wild-type (WT) during normal growth or upon application of UV-C irradiation. Aldehyde profiling in leaves of 24-day-old plants revealed higher accumulation of acrolein, crotonaldehyde, 3Z-hexenal, hexanal and acetaldehyde in aao3 mutants compared with WT leaves. Similarly, higher levels of acrolein, benzaldehyde, crotonaldehyde, propionaldehyde, trans-2-hexenal and acetaldehyde were accumulated in aao3 mutants upon UV-C irradiation. Aldehydes application to plants hastened profuse senescence symptoms and higher accumulation of aldehydes, such as acrolein, benzaldehyde and 4-hydroxy-2-nonenal, in aao3 mutant leaves as compared with WT. The senescence symptoms included greater decrease in chlorophyll content and increase in transcript expression of the early senescence marker genes, Senescence-Related-Gene1, Stay-Green-Protein2 as well as NAC-LIKE, ACTIVATED-BY AP3/P1. Notably, although aao3 had lower ABA content than WT, members of the ABA-responding genes SnRKs were expressed at similar levels in aao3 and WT. Moreover, the other ABA-deficient mutants [aba2 and 9-cis-poxycarotenoid dioxygenase3-2 (nced3-2), that has functional AAO3] exhibited similar aldehydes accumulation and chlorophyll content like WT under normal growth conditions or UV-C irradiation. These results indicate that the absence of AAO3 oxidation activity and not the lower ABA and its associated function is responsible for the earlier senescence symptoms in aao3 mutant.

Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of ß-carotene and, consequently, ß-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing ß-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from ß-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and ß-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from ß-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

Catechins in green tea powder (matcha) are heat-stable scavengers of acrolein, a lipid peroxide-derived reactive carbonyl species.

Lipid peroxidation-derived reactive carbonyl species (RCS) such as acrolein and 4-hydroxynonenal pose health risks. We characterized the RCS-scavenging reactions of tea catechins in an aqueous solution and in baked cake. Acrolein's reaction with each of the major tea catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) resulted in the formation of mono-, di-, and tri-acrolein conjugates of each catechin as revealed by our LC-linear ion trap MS analysis. The formation of the acrolein-conjugates of the four catechins was confirmed in the reaction of acrolein with green tea powder (matcha) extract. The addition of matcha tea powder to cake dough significantly suppressed the accumulation of RCS during cake baking. The mono-acrolein conjugates of the four major catechins were detected in the baked cake. The RCS-scavenging capability of tea catechins offers a new functionality of matcha tea powder, and its heat stability demonstrates the usefulness of matcha as a food additive.

Development of a Novel Detection Method Targeting an Ultrashort 25 bp Sequence Found in Agrobacterium-Mediated Transformed GM Plants.

Agrobacterium-mediated transformation is the most commonly used technique for plant genetic engineering. During the transformation, a T-DNA region, which is flanked by the right border (RB) and the left border, is transferred to plant nuclear chromosomes. Simultaneously, a sequence adjacent to the RB on T-DNA is frequently transferred to plant genomes together with the intentionally introduced recombinant DNA. We developed a novel polymerase chain reaction (PCR)-mediated detection method targeting this region. The conserved sequence of the region found in genetically modified (GM) crops is only 25 bp in length. To detect this ultrashort 25 bp sequence near the RB region, we designed a primer set consisting of a 12-base forward primer and a 13-base reverse primer. The predicted band was detected from GM crops by optimizing the PCR conditions. We used lateral flow DNA chromatography for rapid and inexpensive detection. The developed method would be applicable for screening the GM crops generated by Agrobacterium-mediated transformation.

Reactive Carbonyl Species Mediate Methyl Jasmonate-Induced Stomatal Closure.

Production of reactive oxygen species (ROS) is a key signal event for methyl jasmonate (MeJA)- and abscisic acid (ABA)-induced stomatal closure. We recently showed that reactive carbonyl species (RCS) stimulates stomatal closure as an intermediate downstream of hydrogen peroxide (H2O2) production in the ABA signaling pathway in guard cells of Nicotiana tabacum and Arabidopsis thaliana. In this study, we examined whether RCS functions as an intermediate downstream of H2O2 production in MeJA signaling in guard cells using transgenic tobacco plants overexpressing A. thaliana 2-alkenal reductase (n-alkanal + NAD(P)+ ⇌ 2-alkenal + NAD(P)H + H+) (AER-OE tobacco) and Arabidopsis plants. The stomatal closure induced by MeJA was impaired in the AER-OE tobacco and was inhibited by RCS scavengers, carnosine and pyridoxamine, in the wild-type (WT) tobacco plants and Arabidopsis plants. Application of MeJA significantly induced the accumulation of RCS, including acrolein and 4-hydroxy-(E)-2-nonenal, in the WT tobacco but not in the AER-OE plants. Application of MeJA induced H2O2 production in the WT tobacco and the AER-OE plants and the H2O2 production was not inhibited by the RCS scavengers. These results suggest that RCS functions as an intermediate downstream of ROS production in MeJA signaling and in ABA signaling in guard cells.

Development and Testing of an Individual Kernel Detection System for Genetically Modified Soybean Events in Non-identity-preserved Soybean 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 non-identity-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 herbicide-tolerance, 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.

Correction: Biswas, M.S. et al. Inactivation of Carbonyl-Detoxifying Enzymes by H2O2 Is a Trigger to Increase Carbonyl Load for Initiating Programmed Cell Death in Plants. Antioxidants 2020, 9, 141.

The author wishes to make the following correction to this paper [...].

Inactivation of Carbonyl-Detoxifying Enzymes by H2O2 Is a Trigger to Increase Carbonyl Load for Initiating Programmed Cell Death in Plants.

H2O2-induced programmed cell death (PCD) of tobacco Bright Yellow-2 (BY-2) cells is mediated by reactive carbonyl species (RCS), degradation products of lipid peroxides, which activate caspase-3-like protease (C3LP). Here, we investigated the mechanism of RCS accumulation in the H2O2-induced PCD of BY-2 cells. The following biochemical changes were observed in 10-min response to a lethal dose (1.0 mM) of H2O2, but they did not occur in a sublethal dose (0.5 mM) of H2O2. (1) The C3LP activity was increased twofold. (2) The intracellular levels of RCS, i.e., 4-hydroxy-(E)-hexenal and 4-hydroxy-(E)-nonenal (HNE), were increased 1.2-1.5-fold. (3) The activity of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent carbonyl reductase, scavenging HNE, and n-hexanal was decreased. Specifically, these are the earliest events leading to PCD. The proteasome inhibitor MG132 suppressed the H2O2-induced PCD, indicating that the C3LP activity of the 1 subunit of the 20S proteasome was responsible for PCD. The addition of H2O2 to cell-free protein extract inactivated the carbonyl reductase. Taken together, these results suggest a PCD-triggering mechanism in which H2O2 first inactivates a carbonyl reductase(s), allowing RCS levels to rise, and eventually leads to the activation of the C3LP activity of 20S proteasome. The carbonyl reductase thus acts as an ROS sensor for triggering PCD.

Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction.

Genetically modified (GM) Atlantic salmon, AquAdvantage (AquAd), was the first GM animal approved officially for human consumption. Many countries monitor the use of this product under their GM regulations, but a pragmatic system for AquAd-specific detection is needed. Here, we developed a real-time polymerase chain reaction method with high sensitivity for detection of AquAd in foods. This method showed high specificity for the AquAd transgene and the detection limit was 12.5-25 targeted DNA copies per test reaction. An inter-laboratory study using the method developed demonstrated reproducibility at >0.1% (w/w) AquAd content.

Engineering Yarrowia lipolytica for the utilization of acid whey.

Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted ß-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology.