Sheng Quan

Metabolic variation between japonica and indica rice cultivars as revealed by non-targeted metabolomics

Seed metabolites are critically important both for plant development and human nutrition; however, the natural variation in their levels remains poorly characterized. Here we profiled 121 metabolites in mature seeds of a wide panel Oryza sativa japonica and indica cultivars, revealing correlations between the metabolic phenotype and geographic origin of the rice seeds. Moreover, japonica and indica subspecies differed significantly not only in the relative abundances of metabolites but also in their corresponding metabolic association networks. These findings provide important insights into metabolic adaptation in rice subgroups, bridging the gap between genome and phenome, and facilitating the identification of genetic control of metabolic properties that can serve as a basis for the future improvement of rice quality via metabolic engineering.

One Simple DNA Extraction Device and Its Combination with Modified Visual Loop-Mediated Isothermal Amplification for Rapid On-Field Detection of Genetically Modified Organisms

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

Metabolic map of mature maize kernels

Abstract Metabolites in maize kernels are associated not only with nutritional value but also physiological properties such as maturation, desiccation, and germination. However, comprehensive information concerning the metabolome of maize kernels is limited. In this study, we identified 210 metabolites in mature kernels of 14 representative maize lines using a non-targeted metabolomic profiling approach. Further statistical analysis revealed that 75 metabolites were significantly variable among those tested lines, and certain metabolites out of the detected 210 metabolites played critical roles in distinguishing one line from another. Additionally, metabolite–metabolite correlation analysis dissected key regulatory elements or pathways involved in metabolism of lipids, amino acids and carbohydrates. Furthermore, an integrated metabolic map constructed with transcriptomic, proteomic and metabolic data uncovered characteristic regulatory mechanisms of maize kernel metabolism. Altogether, this work provides new insights into the maize kernel metabolome that would be useful for metabolic engineering and/or molecular breeding to improve maize kernel quality and yield.

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

Comparative metabolomic analysis of wild type and mads3 mutant rice anthers

Rice (Oryza sativa L.) MADS3 transcription factor regulates the homeostasis of reactive oxygen species (ROS) during late anther development, and one MADS3 mutant, mads3-4, has defective anther walls, aborted microspores and complete male sterility. Here, we report the untargeted metabolomic analysis of both wild type and mads3-4 mature anthers. Mutation of MADS3 led to an unbalanced redox status and caused oxidative stress that damages lipid, protein, and DNA. To cope with oxidative stress in mads3-4 anthers, soluble sugars were mobilized and carbohydrate metabolism was shifted to amino acid and nucleic acid metabolism to provide substrates for the biosynthesis of antioxidant proteins and the repair of DNA. Mutation of MADS3 also affected other aspects of rice anther development such as secondary metabolites associated with cuticle, cell wall, and auxin metabolism. Many of the discovered metabolic changes in mads3-4 anthers were corroborated with changes of expression levels of corresponding metabolic pathway genes. Altogether, this comparative metabolomic analysis indicated that MADS3 gene affects rice anther development far beyond the ROS homeostasis regulation.

Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens.

Food allergies cause health risks to susceptible consumers and regulations on labeling of food allergen contents have been implemented in many countries and regions. To achieve timely and accurate food allergen labeling, the development of fast and effective allergen detection methods is very important. Herein, a decaplex polymerase chain reaction (PCR) assay combined with capillary electrophoresis was developed to detect simultaneously 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan. The absolute limit of detection (LODa) of this system is between 2 and 20 copies of haploid genome, and the relative LOD (LODr) is as low as 0.005% (w/w) in simulated food mixtures. The developed assay was subsequently applied to 20 commercial food products and verified the allergen ingredients stated on the labels. Furthermore, results using this decaplex PCR assay was successfully replicated in three other laboratories, demonstrating the repeatability and applicability of this assay in routine analysis of the 10 food allergens.

Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3) is required for anther development and male fertility in rice

Dysfunction of OsGPAT3 leads to disrupted anther lipid metabolism and male sterility in rice.

Molecular characterization of genetically-modified crops: Challenges and strategies

Molecular characterization lays a foundation for safety assessment and subsequent monitoring of genetically modified (GM) crops. Due to the target-specific nature, conventional polymerase chain reaction (PCR)-based methods cannot comprehensively detect unintended gene insertions, let alone unknown GM events. As more and more new developed GM crops including new plant breeding technology (NPBT) generated crops are in the pipeline for commercialization, alternative -omics approaches, particularly next generation sequencing, have been developed for molecular characterization of authorized or unauthorized GM (UGM) crops. This review summarizes first those methods, addresses their challenges, and discusses possible strategies for molecular characterization of engineered crops generated by NPBT, highlighting needs for a global information-sharing database and cost-effective, accurate and comprehensive molecular characterization approaches.

Metabolic changes in transgenic maize mature seeds over-expressing the Aspergillus niger phyA2.

Key messageNon-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing theAspergillus niger phyA2.AbstractGenetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC–MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

The metabolic responses of HepG2 cells to the exposure of mycotoxin deoxynivalenol

As the number of reported deoxynivalenol (DON) contamination incidents increased steadily over the past decades, there has been a widespread interest in understanding the cellular mechanisms of the toxicological effects of DON using in vitro systems and omics technologies. The present investigation was conducted to understand the metabolomic changes in human hepatocellular carcinoma cells (HepG2) exposed to 10 μM DON for short term (4 h) and long term (12 h) periods, using a non-targeted metabolomics approach. Our results revealed a remarkable metabolic shift from short term to long term exposure to DON in HepG2 cells. Our metabolomics data also confirmed the role of DON induced oxidative stress in DON toxicity. Coupled with pattern recognition and pathway analysis, effects of DON on redox homeostasis, energy balance, lipid metabolism, and potential toxicological mechanisms were discussed, which would facilitate further studies on the risk assessment of the dietary mycotoxin DON.